Macroautophagy is a process that leads to the bulk degradation of subcellular constituents by producing autophagosomes/autolysosomes. It is believed that Atg5 (ref. 4) and Atg7 (ref. 5) are essential ...genes for mammalian macroautophagy. Here we show, however, that mouse cells lacking Atg5 or Atg7 can still form autophagosomes/autolysosomes and perform autophagy-mediated protein degradation when subjected to certain stressors. Although lipidation of the microtubule-associated protein light chain 3 (LC3, also known as Map1lc3a) to form LC3-II is generally considered to be a good indicator of macroautophagy, it did not occur during the Atg5/Atg7-independent alternative process of macroautophagy. We also found that this alternative process of macroautophagy was regulated by several autophagic proteins, including Unc-51-like kinase 1 (Ulk1) and beclin 1. Unlike conventional macroautophagy, autophagosomes seemed to be generated in a Rab9-dependent manner by the fusion of isolation membranes with vesicles derived from the trans-Golgi and late endosomes. In vivo, Atg5-independent alternative macroautophagy was detected in several embryonic tissues. It also had a function in clearing mitochondria during erythroid maturation. These results indicate that mammalian macroautophagy can occur through at least two different pathways: an Atg5/Atg7-dependent conventional pathway and an Atg5/Atg7-independent alternative pathway.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of ...the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.
Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on ...atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine C-C motif ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.
Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown ...pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD
)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.
The exploration of oceans using autonomous underwater vehicles (AUVs) is necessary for activities, such as the sustainable management of fishery resources, extraction of seafloor minerals and energy ...resources, and inspection of underwater infrastructure. As the next step in ocean exploration, AUVs are expected to employ end-effectors to make physical contact with seafloor creatures and materials. We propose a scenario for realizing a sampling mission using an AUV that is equipped to sample marine life. In this scenario, the sampling AUV observes the seafloor while concurrently transmitting the observed images to a surface vessel for inspection by the AUV operators. If the received images show an object of interest, the object is selected as a candidate of sampling target by the operators, who send a sampling command to the AUV. After receiving the command, the AUV returns to the target area and attempts to sample it. In this paper, we propose a system for transmitting images of the seafloor as part of the sampling-mission scenario. The proposed image transmission system includes a process for enhancing images of the deep seafloor, a process for selecting interesting images, and processes for compressing and reconstructing images. The image enhancement process resolves imaging problems resulting from light attenuation, such as color attenuation and uneven illumination. The process for selecting interesting images selects those that contain interesting objects, such as marine life. The selection process prevents the transmission of meaningless images that contain only flat sand on the seafloor. The proposed image compression method, which is based on color depth compression, reduces the amount of data. The combined process of selecting an interesting image and compressing it reduces various problems in acoustic communication, such as low information density and data loss. Instead of an overall image, part of an overall image is transmitted by a set of data packet, and each received data packet is reconstructed onboard the vessel. Because of image compression, the colors of a reconstructed image differ from those of an enhanced image. However, the reconstructed image contains similar colors, and the structural similarity index was found to be 91.4% by evaluating images that were subjected to a 4-b color compression. The proposed image transmission system was tested in the Sea of Okhotsk, and these tests were performed four times in different sea areas (minimum depth 380 m, maximum depth 590 m). The results show that the size of the data for a single image was reduced by a factor of 18 using the proposed image compression process, with each image taking 3.7 s to be transmitted via an acoustic modem (20 kb/s). Of the automatically selected images, 63% contained marine life, and the total transmission success rate was 22%.
Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the ...lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.
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•Liver mitochondrial succinylome is quantified in wild-type and Sirt5−/− mice•386 lysine sites on 140 proteins are hypersuccinylated in Sirt5−/− mouse liver•SIRT5 targets enzymes in fatty acid oxidation and ketone body production•Inhibition of HMGCS2 activity by lysine succinylation at K83 and K310
Autophagy has been reported to play a crucial role in the maintenance of intracellular homeostasis, including in pancreatic beta cells. Rubicon, which interacts with the phosphoinositide 3-kinase ...(PI3K) complex, through autophagy-related 14 (ATG14), is among the few autophagy regulators that have been reported to inhibit autophagic flux to date and the deletion of Rubicon has been shown to increase autophagic flux. Based on previous results showing a causal relationship between autophagic dysfunction and pancreatic beta-cell impairment, we hypothesized that the deletion of Rubicon in pancreatic beta cells would improve cell integrity and confer protective effects. To test this hypothesis, we first confirmed that Rubicon knockdown (KD) promoted autophagic flux in βTC3 pancreatic beta-cell line. Next, we generated pancreatic beta-cell-specific Rubicon knockout (βKO) mice, by administering tamoxifen to Rubiconflox/flox:MIP-Cre-ERT mice, which showed normal glucose tolerance and insulin secretion under a normal chow diet, despite successful gene recombination. We also attempted to increase insulin resistance by feeding the mice with a high-fat diet for an additional 2 months to find little differences among the parameters evaluated for glucose metabolism. Finally, severe insulin resistance was induced with insulin receptor antagonist treatment, which resulted in comparable glucose homeostasis measurements between Rubicon βKO and control mice. In summary, these results suggest that in pancreatic beta cells, Rubicon plays a limited role in the maintenance of systemic glucose homeostasis.
Autophagy plays an essential role in preserving cellular homeostasis in pancreatic beta cells. However, the extent of autophagic flux in pancreatic islets induced in various physiological settings ...remains unclear. In this study, we generate transgenic mice expressing pHluorin-LC3-mCherry reporter for monitoring systemic autophagic flux by measuring the pHluorin/mCherry ratio, validating them in the starvation and insulin-deficient model. Our findings reveal that autophagic flux in pancreatic islets enhances after starvation, and suppression of the flux after short-term refeeding needs more prolonged re-starvation in islets than in the other insulin-targeted organs. Furthermore, heterogeneity of autophagic flux in pancreatic beta cells manifests under insulin resistance, and intracellular calcium influx by glucose stimulation increases more in high- than low-autophagic flux beta cells, with differential gene expression, including lipoprotein lipase. Our pHluorin-LC3-mCherry mice enable us to reveal biological insight into heterogeneity in autophagic flux in pancreatic beta cells.
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•Generation of pHluorin-LC3-mCherry mice for evaluating the autophagic flux in vivo•Insulin resistance induces heterogeneity of the autophagic flux in islet cells•The autophagic flux of beta cells is associated with the Ca2+ influx induced by glucose•Regulation of the flux is different between islets and other insulin-targeted organs
Aoyama et al. generate pHluorin-LC3-mCherry mice for evaluating the autophagic flux. Further investigation shows insulin resistance induces heterogeneity of the autophagic flux in pancreatic beta cells, unveiling the association between the flux and the cellular physiological function and different regulations of the flux in beta cells.
CD4
+
T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific ...TCR-transduced CD4
+
T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4
+
T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy.
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