Caspases Switch off the m6A RNA Modification Pathway to Foster the Replication of a Ubiquitous Human Tumor Virus

The methylation of RNA at the N6 position of adenosine (m6A) orchestrates multiple biological processes to control development, differentiation, and cell cycle, as well as various aspects of the virus life cycle. How the m6A RNA modification pathway is regulated to finely tune these processes remains poorly understood. Here, we discovered the m6A reader YTHDF2 as a caspase substrate via proteome-wide prediction, followed by in vitro and in vivo validations. We further demonstrated that cleavage-resistant YTHDF2 blocks, while cleavage-mimicking YTHDF2 fragments promote, the replication of a common human oncogenic virus, Epstein-Barr virus (EBV). Intriguingly, our study revealed a feedback regulation between YTHDF2 and caspase-8 via m6A modification of CASP8 mRNA and YTHDF2 cleavage during EBV replication. Further, we discovered that caspases cleave multiple components within the m6A RNA modification pathway to benefit EBV replication. Our study establishes that caspase disarming of the m6A RNA modification machinery fosters EBV replication

Intracellular cholesterol transport inhibition Impairs autophagy flux by decreasing autophagosome-lysosome fusion

Background: Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. Results: This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome-lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome-lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. Conclusions: Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome-lysosome fusion

Pharmacological inhibition of catalase induces peroxisome leakage and suppression of LPS induced inflammatory response in Raw 264.7 cell.

Peroxisomes are metabolically active organelles which are known to exert anti-inflammatory effects especially associated with the synthesis of mediators of inflammation resolution. However, the role of catalase and effects of peroxisome derived reactive oxygen species (ROS) caused by lipid peroxidation through 4-hydroxy-2-nonenal (4-HNE) on lipopolysaccharide (LPS) mediated inflammatory pathway are largely unknown. Here, we show that inhibition of catalase by 3-aminotriazole (3-AT) results in the generation of peroxisomal ROS, which contribute to leaky peroxisomes in RAW264.7 cells. Leaky peroxisomes cause the release of matrix proteins to the cytosol, which are degraded by ubiquitin proteasome system. Furthermore, 3-AT promotes the formation of 4HNE-IκBα adduct which directly interferes with LPS induced NF-κB activation. Even though, a selective degradation of peroxisome matrix proteins and formation of 4HNE- IκBα adduct are not directly related with each other, both of them are could be the consequences of lipid peroxidation occurring at the peroxisome membrane

Knockdown of PEX16 Induces Autophagic Degradation of Peroxisomes

Peroxisome abundance is regulated by homeostasis between the peroxisomal biogenesis and degradation processes. Peroxin 16 (PEX16) is a peroxisomal protein involved in trafficking membrane proteins for de novo peroxisome biogenesis. The present study demonstrates that PEX16 also modulates peroxisome abundance through pexophagic degradation. PEX16 knockdown in human retinal pigment epithelial-1 cells decreased peroxisome abundance and function, represented by reductions in the expression of peroxisome membrane protein ABCD3 and the levels of cholesterol and plasmalogens, respectively. The activation of pexophagy under PEX16 knockdown was shown by (i) abrogated peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cell lines, and (ii) increased autophagy flux and co-localization of p62—an autophagy adaptor protein—with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. Thus, PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes

Catalase-deficient mice induce aging faster through lysosomal dysfunction

Background: Lysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice. Methods: We performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group. Results: We found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice. Conclusions: This study unveils the new role of catalase and its role in lysosomal function during aging

Ciliogenesis is reciprocally regulated by PPARA and NR1H4/FXR through controlling autophagy in vitro and in vivo

<p>The primary cilia are evolutionarily conserved microtubule-based cellular organelles that perceive metabolic status and thus link the sensory system to cellular signaling pathways. Therefore, ciliogenesis is thought to be tightly linked to autophagy, which is also regulated by nutrient-sensing transcription factors, such as PPARA (peroxisome proliferator activated receptor alpha) and NR1H4/FXR (nuclear receptor subfamily 1, group H, member 4). However, the relationship between these factors and ciliogenesis has not been clearly demonstrated. Here, we present direct evidence for the involvement of macroautophagic/autophagic regulators in controlling ciliogenesis. We showed that activation of PPARA facilitated ciliogenesis independently of cellular nutritional states. Importantly, PPARA-induced ciliogenesis was mediated by controlling autophagy, since either pharmacological or genetic inactivation of autophagy significantly repressed ciliogenesis. Moreover, we showed that pharmacological activator of autophagy, rapamycin, recovered repressed ciliogenesis in <i>ppara⁻^/− </i> cells. Conversely, activation of NR1H4 repressed cilia formation, while knockdown of NR1H4 enhanced ciliogenesis by inducing autophagy. The reciprocal activities of PPARA and NR1H4 in regulating ciliogenesis were highlighted in a condition where de-repressed ciliogenesis by NR1H4 knockdown was further enhanced by PPARA activation. The in vivo roles of PPARA and NR1H4 in regulating ciliogenesis were examined in greater detail in <i>ppara⁻^/⁻ </i> mice. In response to starvation, ciliogenesis was facilitated in wild-type mice via enhanced autophagy in kidney, while <i>ppara⁻^/⁻ </i> mice displayed impaired autophagy and kidney damage resembling ciliopathy. Furthermore, an NR1H4 agonist exacerbated kidney damage associated with starvation in <i>ppara⁻^/⁻ </i> mice. These findings indicate a previously unknown role for PPARA and NR1H4 in regulating the autophagy-ciliogenesis axis in vivo.</p