Regulation of reticulophagy by the N-degron pathway

ABSTRACT

Cellular homeostasis requires selective autophagic degradation of damaged or defective organelles, including the endoplasmic reticulum (ER). Previous studies have shown that specific ER transmembrane receptors recruit LC3 on autophagic membranes by using LC3-interacting domains. In this study, we showed that the N-degron pathway mediates ubiquitin (Ub)-dependent reticulophagy. During this 2-step process, the ER transmembrane E3 ligase TRIM13 undergoes auto-ubiquitination via lysine 63 (K63) linkage chains and acts as a ligand for the autophagic receptor SQSTM1/p62 (sequestosome 1). In parallel, ER-residing molecular chaperones, such as HSPA5/GRP78/BiP, are relocated to the cytosol and conjugated with the amino acid L-arginine (Arg) at the N-termini by ATE1 (arginyltransferase 1). The resulting N-terminal Arg (Nt-Arg) binds the ZZ domain of SQSTM1, inducing oligomerization of SQSTM1-TRIM13 complexes and facilitating recruitment of LC3 on phagophores to the sites of reticulophagy. We developed small molecule ligands to the SQSTM1 ZZ domain and demonstrate that these chemical mimics of Nt-Arg facilitate reticulophagy and autophagic protein quality control of misfolded aggregates in the ER.     

Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant

Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum(ER)fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells.However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3-or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.     

Restoration of ER proteostasis attenuates remote apoptotic cell death after spinal cord injury by reducing autophagosome overload

The pathogenic mechanisms that underlie the progression of remote degeneration after spinal cord injury (SCI) are not fully understood. In this study, we examined the relationship between endoplasmic reticulum (ER) stress and macroautophagy, hereafter autophagy, and its contribution to the secondary damage and outcomes that are associated with remote degeneration after SCI. Using a rat model of spinal cord hemisection at the cervical level, we measured ER stress and autophagy markers in the axotomized neurons of the red nucleus (RN). In SCI animals, mRNA and protein levels of markers of ER stress, such as GRP78, CHOP, and GADD34, increased 1 day after the injury, peaking on Day 5. Notably, in SCI animals, the increase of ER stress markers correlated with a blockade in autophagic flux, as evidenced by the increase in microtubule-associated protein 2 light chain 3 (LC3-II) and p62/SQSTM1 (p62) and the decline in LAMP1 and LAMP2 levels. After injury, treatment with guanabenz protected neurons from UPR failure and increased lysosomes biogenesis, unblocking autophagic flux. These effects correlated with greater activation of TFEB and improved neuronal survival and functional recovery—effects that persisted after suspension of the treatment. Collectively, our results demonstrate that in remote secondary damage, impairments in autophagic flux are intertwined with ER stress, an association that contributes to the apoptotic cell death and functional damage that are observed after SCI.Subject terms: Cell death in the nervous system, Neurodegeneration, Molecular neuroscience     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

SQSTM1/p62 (sequestosome 1) senses cellular ubiquitin stress through E2-mediated ubiquitination

The alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. We recently reported that the macroautophagy/autophagy receptor SQSTM1/p62, functions as a novel Ub sensor to activate autophagy upon Ub⁺ stress (upregulation of the Ub level). First, SQSTM1 was found to undergo extensive ubiquitination and activate autophagy under Ub⁺ stress induced by prolonged Bortezomib (BTZ) treatment, Ub overexpression or by heat shock. Mechanistically, Ubiquitination of SQSTM1 disrupts its dimerization of the UBA domain, switching it from an auto-inhibitory conformation to recognize poly-ubiquitinated cargoes, promoting autophagic flux. Interestingly, Ub⁺ stress-responsive SQSTM1 ubiquitination is mediated by Ub conjugating enzymes, UBE2D2/3, in a unique E2-dependent manner. Our work has thus revealed a novel mechanism for how SQSTM1 senses cellular Ub stress conditions and regulates selective autophagy in response to diverse intrinsic or extrinsic challenges.     

CCPG1 is a noncanonical autophagy cargo receptor essential for reticulophagy and pancreatic ER proteostasis

Reticulophagy is the conserved macroautophagic/autophagic degradation of the endoplasmic reticulum (ER) in response to ER stress or general nutrient deprivation. Sequestration of the ER by phagophores plays an important role in regulating ER size and homeostasis. In their recent work, Smith et al. have discovered that the ER-localized protein CCPG1 is a novel mammalian reticulophagy receptor that interacts with core autophagy machinery components—LC3, GABARAP and RB1CC1—and regulates reticulophagy.     

CALCOCO1 is a soluble reticulophagy receptor

ABSTRACT

The endoplasmic reticulum (ER) is the largest membrane-bound organelle in eukaryotic cells and plays critical roles in diverse processes in metabolism, signaling and intracellular organization. In response to stress stimuli such as nutrient deprivation, accumulation of misfolded proteins or exposure to chemicals, the ER increases in size through upregulated synthesis of its components to counteract the stress. To restore physiological size, the excess ER components are continuously dismantled and degraded by reticulophagy, a form of autophagy that targets, via adaptor molecules called reticulophagy receptors, specific ER portions to the lysosome for degradation. Previous studies have identified several ER resident proteins as reticulophagy receptors. In a recent study, we identified CALCOCO1 as a soluble reticulophagy receptor for the degradation of tubular ER in response to proteotoxic and starvation-induced stress. On the ER membrane, CALCOCO1 interacts with VAPA and VAPB via a FFAT-like motif and recruits autophagy machinery by binding directly to Atg8-family proteins via LIR and UDS interacting region (UIR) motifs acting co-dependently. Depletion of CALCOCO1 in cultured cells led to an impaired ER degradation during stress.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Vitamin D Inhibits Human Immunodeficiency Virus Type 1 and Mycobacterium tuberculosis Infection in Macrophages through the Induction of Autophagy

Low vitamin D levels in human immunodeficiency virus type-1 (HIV) infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We have previously shown that 1α,25-dihydroxycholecalciferol (1,25D3), the active form of vitamin D, inhibits HIV replication in human macrophages through the induction of autophagy. In this study, we report that physiological concentrations of 1,25D3 induce the production of the human cathelicidin microbial peptide (CAMP) and autophagic flux in HIV and M. tuberculosis co-infected human macrophages which inhibits mycobacterial growth and the replication of HIV. Using RNA interference for Beclin-1 and the autophagy-related 5 homologue, combined with the chemical inhibitors of autophagic flux, bafilomycin A₁, an inhibitor of autophagosome-lysosome fusion and subsequent acidification, and SID 26681509 an inhibitor of the lysosome hydrolase cathepsin L, we show that the 1,25D3-mediated inhibition of HIV replication and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Moreover, through the use of RNA interference for CAMP, we demonstrate that cathelicidin is essential for the 1,25D3 induced autophagic flux and inhibition of HIV replication and mycobacterial growth. The present findings provide a biological explanation for the benefits and importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.     

Autophagic flux,supported by toll-like receptor 2 activity,defends against the carcinogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the most common primary malignant liver tumor, is the third leading cause of cancer deaths. The pathogenesis of HCC is closely associated with chronic liver inflammation fired by a variety of stimulates such as virus infection and metabolic stress. Recent work indicates that autophagy, a homeostatic self-degradation process, which decides cell survival or death upon stress, acts as an effector machinery of immune systems in defending microbial invasion and carcinogenesis. SQSTM1 is a selective target and receptor of autophagy, and the protein content of SQSTM1 reflects the level of autophagic flux in cells. Through degrading SQSTM1, decreasing SQSTM1 aggregates, and therefore interrupting the positive feedback between SQSTM1 aggregates and ROS production, autophagy plays a protective role against hepatocellular carcinoma. Indeed, our studies indicate that toll-like receptor 2 (TLR2)-mediated immune activities in the genotoxic carcinogen diethylnitrosamine (DEN)-injured liver tissue provide essential nutrient stimulates to induce intracellular senescence, which can ensure the activation and maturation of autophagy in liver cells. Loss of TLR2-mediated immune activity and senescence leads to the attenuation of autophagic flux, which cannot eliminate SQSTM1 aggregates, ROS accumulation, and DNA damage, and facilitates the development and progression of HCC.     

Increased transport of acetyl‐CoA into the endoplasmic reticulum causes a progeria‐like phenotype

The membrane transporter AT‐1/SLC33A1 translocates cytosolic acetyl‐CoA into the lumen of the endoplasmic reticulum (ER), participating in quality control mechanisms within the secretory pathway. Mutations and duplication events in AT‐1/SLC33A1 are highly pleiotropic and have been linked to diseases such as spastic paraplegia, developmental delay, autism spectrum disorder, intellectual disability, propensity to seizures, and dysmorphism. Despite these known associations, the biology of this key transporter is only beginning to be uncovered. Here, we show that systemic overexpression of AT‐1 in the mouse leads to a segmental form of progeria with dysmorphism and metabolic alterations. The phenotype includes delayed growth, short lifespan, alopecia, skin lesions, rectal prolapse, osteoporosis, cardiomegaly, muscle atrophy, reduced fertility, and anemia. In terms of homeostasis, the AT‐1 overexpressing mouse displays hypocholesterolemia, altered glycemia, and increased indices of systemic inflammation. Mechanistically, the phenotype is caused by a block in Atg9a‐Fam134b‐LC3β and Atg9a‐Sec62‐LC3β interactions, and defective reticulophagy, the autophagic recycling of the ER. Inhibition of ATase1/ATase2 acetyltransferase enzymes downstream of AT‐1 restores reticulophagy and rescues the phenotype of the animals. These data suggest that inappropriately elevated acetyl‐CoA flux into the ER directly induces defects in autophagy and recycling of subcellular structures and that this diversion of acetyl‐CoA from cytosol to ER is causal in the progeria phenotype. Collectively, these data establish the cytosol‐to‐ER flux of acetyl‐CoA as a novel event that dictates the pace of aging phenotypes and identify intracellular acetyl‐CoA‐dependent homeostatic mechanisms linked to metabolism and inflammation.     

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.     

Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway

Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ?dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival and proliferation.