VCP/p97 modulates PtdIns3P production and autophagy initiation
9
Citation
1
Reference
10
Related Paper
Citation Trend
Abstract:
VCP/p97 is an essential multifunctional protein implicated in a plethora of intracellular quality control systems, and abnormal function of VCP is the underlying cause of several neurodegenerative disorders. We reported that VCP regulates the levels of the macroautophagy/autophagy-inducing lipid phosphatidylinositol-3-phosphate (PtdIns3P) by modulating the activity of the BECN1 (beclin 1)-containing phosphatidylinositol 3-kinase (PtdIns3K) complex. VCP stimulates the deubiquitinase activity of ATXN3 (ataxin 3) to stabilize BECN1 protein levels and also interacts with and promotes the assembly and kinase activity of the PtdIns3K complex. Acute inhibition of VCP activity impairs autophagy induction, demonstrated by a diminished PtdIns3P production and decreased recruitment of early autophagy markers WIPI2 and ATG16L1. Thus, VCP promotes autophagosome biogenesis, in addition to its previously described role in autophagosome maturation.Keywords:
BECN1
Autophagosome
ULK1
ATG16L1
BAG3
Organelle biogenesis
VCP/p97 is an essential multifunctional protein implicated in a plethora of intracellular quality control systems, and abnormal function of VCP is the underlying cause of several neurodegenerative disorders. We reported that VCP regulates the levels of the macroautophagy/autophagy-inducing lipid phosphatidylinositol-3-phosphate (PtdIns3P) by modulating the activity of the BECN1 (beclin 1)-containing phosphatidylinositol 3-kinase (PtdIns3K) complex. VCP stimulates the deubiquitinase activity of ATXN3 (ataxin 3) to stabilize BECN1 protein levels and also interacts with and promotes the assembly and kinase activity of the PtdIns3K complex. Acute inhibition of VCP activity impairs autophagy induction, demonstrated by a diminished PtdIns3P production and decreased recruitment of early autophagy markers WIPI2 and ATG16L1. Thus, VCP promotes autophagosome biogenesis, in addition to its previously described role in autophagosome maturation.
BECN1
Autophagosome
ULK1
ATG16L1
BAG3
Organelle biogenesis
Cite
Citations (9)
Autophagy is an evolutionary conserved, degradative process from single-cell eukaryotes, such as
BECN1
ULK1
Autophagy-related protein 13
BAG3
TOR signaling
ATG16L1
ATG8
Cite
Citations (109)
Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.
Autophagy-related protein 13
ATG8
ULK1
BAG3
BECN1
Cite
Citations (51)
VCP, a conserved ATPase, is involved in several cellular processes, and mutations in this protein are associated with various diseases. VCP also plays a role in autophagosome maturation. However, because a deficiency in autophagosome maturation presents a readily observable phenotype, other roles of VCP in autophagy regulation, in particular in the initial steps of autophagosome formation, may have been overlooked. In a recently published paper, using small-molecule inhibitors, Hill et al. showed that VCP regulates autophagy initiation through both stabilization of BECN1 and enhancement of phosphati-dylinositol 3-kinase (PtdIns3K) complex assembly.
BECN1
Autophagosome
BAG3
ULK1
Autophagy-related protein 13
Cite
Citations (5)
Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions.
ULK1
BAG3
ATG16L1
Autophagy-related protein 13
Cite
Citations (36)
ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.
ULK1
Autophagy-related protein 13
BECN1
Autophagosome
BAG3
Cite
Citations (283)
Adenosine monophosphate-activated protein kinase (AMPK) is a significant energy sensor in the maintenance of cellular energy homeostasis. Autophagy is a highly conserved catabolic process that involves an intracellular degradation system in which cytoplasmic components, such as protein aggregates, organelles, and other macromolecules, are directed to the lysosome through the self-degradative process to maintain cellular homeostasis. Given the triggered autophagy process in various situations including the nutrient deficit, AMPK is potentially linked with different stages of autophagy. Above all, AMPK increases ULK1 activity by directly phosphorylating Ser467, Ser555, Thr574, and Ser637 at least four sites, which increases the recruitment of autophagy-relevant proteins (ATG proteins) to the membrane domains which affects autophagy at the initiation stage. Secondly, AMPK inhibits VPS34 complexes that do not contain pro-autophagic factors and are thus involved in isolation membrane forming processes, by direct phosphorylation of VPS34 on Thr163 and Ser165. After phosphorylation, AMPK can govern autophagosome formation through recruiting downstream autophagy-related proteins to the autophagosome formation site. Finally, the AMPK-SIRT1 signaling pathway can be activated by upregulating the transcription of autophagy-related genes, thereby enhancing autophagosome-lysosome fusion. This review provides an introduction to the role of AMPK in different stages of autophagy.
Autophagosome
ULK1
ATG16L1
BAG3
Cite
Citations (84)
ABSTRACTMacroautophagy/autophagy is a tightly regulated cellular process integral to homeostasis and innate immunity. As such, dysregulation of autophagy is associated with cancer, neurodegenerative disorders, and infectious diseases. While numerous factors that promote autophagy have been characterized, the key mechanisms that prevent excessive autophagy are less well understood. Here, we identify CSNK2/CK2 (casein kinase 2) as a negative regulator of autophagy. Pharmacological inhibition of CSNK2 activity or siRNA-mediated depletion of CSNK2 increased basal autophagic flux in cell lines and primary human lung cells. Vice versa, ectopic expression of CSNK2 reduced autophagic flux. Mechanistically, CSNK2 interacted with the FLN (filamin)-NHL domain-containing tripartite motif (TRIM) family members TRIM2, TRIM3 and TRIM71. Our data show that recruitment of CSNK2 to the C-terminal NHL domain of TRIM3 lead to its robust phosphorylation at serine 661 by CSNK2. A phosphorylation-defective mutant of TRIM3 was unable to reduce autophagosome numbers indicating that phosphorylation by CSNK2 is required for TRIM-mediated autophagy inhibition. All three TRIMs facilitated inactivation of the ULK1-BECN1 autophagy initiation complex by facilitating ULK1 serine 757 phosphorylation. Inhibition of CSNK2 promoted autophagy upon influenza A virus (IAV) and measles virus (MeV) infection. In line with this, targeting of CSNK2 or depletion of TRIM2, TRIM3 or TRIM71 enhanced autophagy-dependent restriction of IAV, MeV and human immunodeficiency virus 1 (HIV-1). Thus, our results identify the CSNK2-TRIM2, -TRIM3, -TRIM71 axis as a key regulatory pathway that limits autophagy. Targeting this axis may allow for therapeutic induction of autophagy against viral infections and in diseases associated with dysregulated autophagy.KEYWORDS: Autophagycasein kinasetripartite motif proteinsvirusDisclaimerAs a service to authors and researchers we are providing this version of an accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proofs will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to these versions also. ACKNOWLEDGEMENTSWe thank Daniela Krnavek, Martha Mayer, Kerstin Regensburger, Regina Burger, Jana-Romana Fischer, Birgit Ott, Meta Volcic and Fabian Zech for assistance and discussions.DISCLOSURE STATEMENTThe authors declare no competing interests.Supplementary materialSupplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2281128DATA AVAILABILITY STATEMENTThe mass spectrometry data generated during this study was deposited at Massive ProteomeExchange with the accession number MSV000091956.Table 1. Oligonucleotides for cloning.Download CSVDisplay TableFigure 1. CSNK2 inhibition induces autophagy. (A) Autophagosome levels in HeLa GL cells treated with kinase inhibitors (10 µM) and bafilomycin A1 (BafA1, co-treatment, 250 nM) as assessed 4 h post stimulation by flow cytometry (eGFP-LC3B MFI). Selected agents are annotated, full list in Table S1. Lines represent the mean ± SEM, n=3. (B) Cell metabolic activity in samples from (A) was assessed via MTT assay 4 h post-treatment depicted as a heatmap of means as indicated, n=3. (C) eGFP-LC3B puncta quantification in HeLa GL cells treated with increasing concentrations of CX-4945 (3.12-25 µM) for 24 h as indicated. Treatment with torin-1 (5 µM) and bafilomycin A1 (BafA1, 250 nM) for 24 h served as control. Lines represent the mean ± SEM, n=29-33 (individual cells). (D) Representative fluorescence confocal laser scanning fluorescence microscopy images from (C). eGFP-LC3B (green), nuclei (DAPI, blue). Scale bar: 25 µm. (E) Autophagosome levels (eGFP-LC3B MFI) in HeLa GL cells treated with increasing concentrations of CX-4945 or GO289 (0.25-32 µM), co-treated with bafilomycin A1 (BafA1, 250 nM) for 4 h. Dots represent the mean ± SEM, n=3. EC50 of CX-4945 and GO289 are indicated next to the respective legend. Student's t-test with Welch correction. *, p<0.05; ***, p<0.001.Display full sizeFigure 2. CSNK2 activity reduces autophagy. (A) Autophagosome levels (eGFP-LC3B MFI) in HEK293T GL cells depleted by siRNA of CSNK2 or MTOR quantified 48 h post transfection with/without co-treatment of bafilomycin A1 (BafA1, 250 nM, 4 h) using flow cytometry. Bars represent mean ± SEM, n=3. (B) Autophagosome levels (eGFP-LC3B MFI) in HEK293T GL cells transiently expressing indicated CSNK2 subunits or the whole complex as assessed by flow cytometry. (right panel) Representative immunoblot showing expression of the HA or V5 tagged CSNK2 subunits. Mock, no transfection. (C) Quantification of LC3-II:LC3-I levels relative to the untreated control (mock) in whole cell lysates of normal human lung fibroblasts (NHLF) treated with CX-4945 (50-0.39 µM) for 24 h as indicated. Bars represent mean ± SEM, n=3, (independent experiments). (bottom panel) Representative immunoblots. (D) Quantification of SQSTM1:GAPDH in whole cell lysates of normal human lung fibroblasts (NHLF) treated with CX-4945 (50-0.39 µM) for 24 h as indicated, relative to the control. (bottom panel) Representative immunoblots. Torin-1 (5 µM) and bafilomycin A1 (BafA1, 250 nM) served as positive controls. Bars represent mean ± SEM, n=3 (independent experiments). Student's t-test with Welch correction. *, p<0.05; **, p<0.01; ***, p<0.001.Display full sizeFigure 3. TRIM2, TRIM3 and TRIM71 inhibit autophagy initiation. (A) Schematic depiction of the domain organization of the FLN-NHL TRIMs (TRIM2, TRIM3, TRIM71) and closely related TRIM proteins. TRIM, tripartite motif. (B) Quantification of autophagosome levels in HEK293T GL cells transiently expressing the indicated FLAG-tagged TRIMs at 48 h post transfection by flow cytometry. Lines represent mean ± SEM, n=3. (right panel) Representative immunoblot of whole cell lysates. (C) Representative confocal laser scanning fluorescence microscopy images of HeLa GL cells transiently expressing indicated TRIMs (48 h) or treated with chloroquine (CQ, 10 µM, overnight). TRIMs (FLAG, red), eGFP-LC3B (green), nuclei (DAPI, blue). Scale bar: 10 µm. (D) Quantification of eGFP-puncta area per cell in the images from (C). Lines represent mean ± SEM, n=61-139 (individual cells). (E) Representative immunoblots of whole cell lysates of HEK293T cells transiently expressing indicated TRIMs (48 h post transfection). Blots were stained with anti-SQSTM1 and anti-GAPDH. Torin-1 (5 µM) and bafilomycin A1 (BafA1, 250 nM) treatment were used as positive controls. (F) Quantification of the immunoblotting data of (E). Lines represent mean ± SEM, n=3. (G) Representative confocal laser scanning fluorescence microscopy images of HeLa GL cells (48 h post transfection) depleted of the indicated TRIMs by siRNA or treated with chloroquine (CQ, 10 µM, overnight). eGFP-LC3B (green), nuclei (DAPI, blue). Scale bar: 10 µm. (H) Quantification of the eGFP-puncta area per cell in the images from (F), Lines represent mean ± SEM, n=61–117 (individual cells). (I) Representative immunoblots of whole cell lysates of HDF cells depleted of the indicated TRIMs by siRNA at 40 h post transfection. Blots were stained with anti-p-ULK1 (S757), anti-ULK1, anti-p-BECN1 (Ser15), anti-BECN1 and anti-ACTB. (J) Quantification of the band intensities of p-ULK1 (Ser757) to total ULK1 of the immunoblotting data of (I). Bars represent mean ± SEM, n=3. (K) Representative immunoblots of whole cell lysates of HEK293T cells that transiently expressed empty vector or TRIM3 at 40 h post transfection. Blots were stained with anti-p-ULK1 (Ser757), anti-ULK1, anti-p-BECN1 (Ser15), anti-BECN1, anti-FLAG and anti-ACTB. (L) Quantification of the band intensities of p-ULK1 (Ser757):ULK1 levels of the immunoblotting data of (K) at highest TRIM3 transfection levels. Bars represent mean ± SEM, n=5. Student's t-test with Welch correction. *, p<0.05; **, p<0.01; ***, p<0.001.Display full sizeFigure 4. CSNK2 interacts with FLN-NHL TRIMs. (A) Representative Immunoblots of an anti-V5 immunoprecipitation of whole cell lysates of HEK293T cells transiently expressing FLAG-tagged TRIM2, TRIM3 or TRIM71, and V5-tagged CSNK2 subunits as indicated. Blots were stained with anti-FLAG, anti-HA and anti-ACTB. WCL, whole cell lysate. (B) Scatter Plot depicting enrichment of proteins co-purifying FLAG-tagged RING deleted mutants of TRIM2 and TRIM3 of a large-scale affinity isolation in HEK293T cells. Enrichments calculated relative to the vector control as label free quantification signal. CSNK2 subunits are highlighted in red. (C) Co-enrichment of the CSNK2 subunits with RING deleted versions of TRIM2 and TRIM3 extracted from the data in (B). Log2 of the label free quantification signal is shown as a heatmap as indicated. n.d., not detected. (D) Representative confocal microscopy images showing the colocalization of overexpressed CSNK2-V5 with each FLAG-tagged TRIM2, TRIM3 and TRIM71 in HeLa cells compared to the single stained mock controls by PLA (red). Nuclei, DAPI (blue). Scale bar: 25 µm. (right panel) Quantification of the PLA signal as pixel per cell. Lines represent mean ± SEM, n=22-47 (individual cells). (E) Representative confocal microscopy images showing the colocalization of endogenous CSNK2A1 and TRIM3 in NHLF cells compared to the single stained mock controls by PLA (red). Nuclei, DAPI (blue). Scale bar: 10 µm. (right panel) Quantification of the PLA signal as puncta per cell. Lines represent mean ± SEM, n=41-94 (individual cells). Student's t-test with Welch correction. ***, p<0.001.Display full sizeFigure 5. TRIM3 mediated autophagy modulation is dependent on its NHL domain. (A) Schematic depiction of the expression patterns of TRIM2, TRIM3 and TRIM71 across the human body. See Figure S5A. (B) Quantification of autophagosome levels (eGFP-LC3B MFI) in HEK293T GL cells transiently expressing TRIM3 or a vector control. 48 h post transfection, cells were treated with increasing concentration of CX-4945 (4 h) and autophagosome levels (eGFP-LC3B MFI) were quantified using flow cytometry. Treatment with bafilomycin A1 (BafA1, 250 nM) was used as a positive control. Lines represent mean ± SEM, n=3. (C) Schematic depiction of the domain organization of TRIM3 and the ΔRING and ΔNHL truncation mutants. (D) Anti-FLAG immunoprecipitation (IP) in whole cell lysates of HEK293T cells transiently expressing HA-tagged CSNK2A2 and indicated FLAG-tagged TRIM3 truncation mutants. 48 h post transfection whole cell lysates (WCLs) and precipitates were analyzed by immunoblotting. Blots were stained with anti-HA, anti-FLAG and anti-ACTB. (E) Quantification of autophagosome levels (eGFP-LC3B MFI, 48 h post transfection) in HEK293T GL cells transiently expressing indicated TRIM3 truncation mutants. Lines represent mean ± SEM, n=3. (F) Representative confocal laser scanning fluorescence microscopy images of HeLa GL cells transiently expressing indicated FLAG-tagged TRIM3 truncation mutants (48 h post transfection). TRIMs (FLAG, red), eGFP-LC3B (green), nuclei (DAPI, blue). Scale bar: 10 µm. (G) Quantification of the eGFP-puncta area per cell in the images from (F). Lines represent mean ± SEM, n=42-57 (individual cells). Student's t-test with Welch correction. *, p<0.05; ***, p<0.001.Display full sizeFigure 6. CSNK2 phosphorylates TRIM3 at S661 to inhibit autophagy. (A) Ubiquitination status of indicated HA-tagged CSNK2 subunits purified by anti-HA immunoprecipitation from whole lysates of transfected HEK293T cells 40 h post transfection. IPs and whole cell lysates (WCL) were analyzed by immunoblotting. Blots were stained with anti-ub (ubiquitin), anti-FLAG and anti-HA. (B) Quantification of CSNK2 activity in whole cell lysates of HEK293T cells transiently expressing indicated proteins and/or treated with CX-4945 (10 µM) by ELISA. Data are relative to the vector control. Lines represent mean ± SEM, n=3. (C and D) Analysis of the phosphorylation status of TRIM3 in HEK293T cells transiently expressing TRIM3 or a vector control by (C) PhosTag acrylamide gel analysis (the white arrow indicates the shifted phosphorylated TRIM3) and (D) enrichment of the FLAG-tagged TRIM3 and immunoblot analysis using phosphor-specific antibodies. Blots were stained with anti-FLAG, anti-p-Ser and anti-p-Thr/Tyr. CalA, Calyculin A. (E) Analysis of the phosphorylation status of TRIM3 purified from HEK293T cells expressing TRIM3 or a vector control and depleted of the indicated proteins using siRNAs targeting individual components of the CSNK2 complex (as indicated) or the whole complex (si.CSNK2) as assessed by immunoblotting. Blots were stained with anti-FLAG and anti-pan-p-Ser. (F) Schematic depiction of the domain organization of the indicated TRIMs and the position and sequence context of the predicted putative CSNK2 phosphorylation sites. (G) Exemplary representative laser scanning confocal microscopy images of HEK293T GL cells transiently expressing the indicated TRIM3 mutants or a vector control 48 h post transfection. TRIMs (FLAG, red), eGFP-LC3B (green), nuclei (DAPI, blue). Scale bar: 10 µm. (H) eGFP-puncta area per cell from the pictures in (G) was quantified. Lines represent mean ± SEM, n=59-112 (individual cells) (I) Quantification of autophagosome levels (eGFP-LC3B MFI, 48 h post transfection) in HEK293T GL cells transiently expressing indicated TRIM3 mutants or a vector control. Bafilomycin A1 (BafA1, 250 nM, overnight) was used a positive control. Lines represent mean ± SEM, n=3. (J) Analysis of the phosphorylation status of TRIM3 WT and TRIM3 S661A purified by anti-FLAG IP from whole cell lysates of HEK293T cells transiently expressing indicated TRIM3 mutants or a vector control and analyzed by immunoblots. Student's t-test with Welch correction. *, p<0.05; **, p<0.01; ***, p<0.001.Display full sizeFigure 7. Pharmacological targeting of the CSNK2-TRIM2, -TRIM3, -TRIM71 axis restricts virus growth. (A and B) Quantification of autophagosome levels (eGFP-LC3B MFI) in HeLa GL cells, with and without CX-4945 (1 µM, 1 h) treatment were infected with (A) influenza A virus (IAV, MOI 5, 5 h) or (B) measles virus (MeV, MOI 1, 24 h) by flow cytometry. Torin-1 (1 µM, 4 h) and bafilomycin A1 (BafA1, 250 nM, 4 h) treatment was used as controls. Lines represent mean ± SEM, n=3-6. (C and D) A549 WT and A549 ATG5 KO cells were treated with CX-4945 (25 µM, 50 µM, 1 h) or left untreated and infected with (C) IAV-GFP or (D) MeV-GFP. Infection kinetics were monitored via fluorescence microscopy for 30 h (IAV-GFP) or 40 h (MeV-GFP) by quantifying GFP+ cell count every 3h and normalized to maximum infection (100 %). Representative fluorescence microscopy images are shown of time-point 24 h. Infected cells (GFP, green). Scale bars: 100 µm. Dots represent the mean ± SEM, n=3. (E and F) Relative comparison of (E) IAV-GFP or (F) MeV-GFP replication in A549 WT and A549 ATG5 KO cells using area under the curve analysis of the data in (C) and (D). Bars represent mean ± SEM, n=3. (G and H) A549 WT and A549 ATG5 KO cells were treated with CX-4945 (6.25 µM – 25 µM, 2 h) or left untreated, and infected with (G) IAV-GFP or (H) MeV-GFP. Supernatants were collected 24 h (IAV-GFP) or 48 h (MeV-GFP) post infection. Infectious virus titers from A549 cells treated with IAV-GFP infected supernatants (G) or from Vero cells treated with MeV-GFP infected supernatants (H). TCID50 (IAV-GFP) and FFU/mL (MeV-GFP) were determined at 72 h post infection. Relative infectious virus titers are normalized to the mock control (DMSO). Bars represent mean ± SEM, n=3 (IAV) or 6 (MeV). (I) Infectious virus yields of HIV-1 NL4-3 proviral DNA transfected HEK293T WT or HEK293T ATG5 KO cells treated with increasing concentrations of CX-4945 (1.56–12.5 µM) and quantified by TZM-bl reporter assay 24 h post transfection. Relative infectious virus yield is normalized to the untreated control (mock). Treatment with torin-1 (1 µM) was used as a control. Bars represent mean ± SEM, n=3. (J) Quantification of infectious virus yield of HIV-1 CH077 in TZM-bl cells depleted of TRIM2, TRIM3 and TRIM71 by siRNA and infected with CH077 HIV-1 one day post transfection. Three days post infection, the infectious virus yield in the supernatant was determined by TZM-bl reporter assay. Relative infectious virus yield is normalized to the non-targeting (NT) control (100 %). Lines represent mean ± SEM, n=3 (technical replicates). Student's t-test with Welch correction or two-way ANOVA (G, H). *, p<0.05; **, p<0.01; ***, p<0.001.Display full sizeAdditional informationFundingThis study was supported by DFG grants to F.K. and K.M.J.S. (CRC 1279 and SPP1923), the BMBF to K.M.J.S. (IMMUNOMOD-01KI2014), and the U.S. National Institutes of Health (NIH) grants R01 AI148534 and R37 AI087846 (to M.U.G). L.K., H.H., V.H. and D.F. are part of the International Graduate School for Molecular Medicine, Ulm (IGradU).
BECN1
BAG3
ULK1
Sequestosome 1
ATG16L1
Autophagosome
Cite
Citations (8)
Selectivity of autophagy is achieved by target recognition; however, the number of autophagy receptors identified so far is limited. In this study we demonstrate that a subset of tripartite motif (TRIM) proteins mediate selective autophagy of key regulators of inflammatory signaling. MEFV/TRIM20, and TRIM21 act as autophagic receptors recognizing their cognate targets and delivering them for autophagic degradation. MEFV recognizes the inflammasome components NLRP3, CASP1 and NLRP1, whereas TRIM21 specifically recognizes the activated, dimeric from of IRF3 inducing type I interferon gene expression. MEFV and TRIM21 have a second activity, whereby they act not only as receptors but also recruit and organize key components of autophagic machinery consisting of ULK1, BECN1, ATG16L1, and mammalian homologs of Atg8, with a preference for GABARAP. MEFV capacity to organize the autophagy apparatus is affected by common mutations causing familial Mediterranean fever. These findings reveal a general mode of action of TRIMs as autophagic receptor-regulators performing a highly-selective type of autophagy (precision autophagy), with MEFV specializing in the suppression of inflammasome and CASP1 activation engendering IL1B/interleukin-1β production and implicated in the form of cell death termed pyroptosis, whereas TRIM21 dampens type I interferon responses.
ATG16L1
ULK1
Pyroptosis
MEFV
IRF3
BECN1
RIG-I
Pyrin domain
BAG3
Sequestosome 1
Cite
Citations (106)
Autophagy is a dynamic and self-limiting process. The amplitude and duration of this process need to be properly controlled to maintain cell homeostasis, and excessive or insufficient autophagy activity could each lead to disease states. Compared to our understanding of the molecular mechanisms of autophagy induction, little is known about how the autophagy process is turned off after its activation. We recently identified KLHL20 as a key regulator of autophagy termination. By functioning as a substrate-binding subunit of CUL3 ubiquitin ligase, KLHL20 targets the activated ULK1 and phagophore-residing PIK3C3/VPS34 and BECN1 for ubiquitination and proteasomal degradation, which in turn triggers a destabilization of their complex components ATG13 and ATG14. These hierarchical degradation events cause the exhaustion of the autophagic pool of ULK1 and PIK3C3/VPS34 complexes, thereby preventing persistent and excessive autophagy activity. Impairment of KLHL20-dependent feedback regulation of autophagy enhances cell death under prolonged starvation and aggravates muscle atrophy in diabetic mice, which highlights the pathophysiological significance of this autophagy termination mechanism in cell survival and tissue homeostasis. Modulation of this autophagy termination pathway may be effective for treating diseases associated with deregulation of autophagy activity.
ULK1
BECN1
BAG3
Protein Degradation
Cite
Citations (13)