Chen, h. | Search bioRxiv

bioRxiv2024

Small-molecule Activation of TFEB Alleviates Niemann-Pick Disease Type C via Promoting Lysosomal Exocytosis and Biogenesis

Niemann-Pick disease type C (NPC) is a devastating lysosomal storage disease characterized by abnormal cholesterol accumulation in lysosomes. Currently, there is no treatment for NPC. Transcription factor EB (TFEB), a member of the microphthalmia transcription factors (MiTF), has emerged as a master regulator of lysosomal function and promoted the clearance of substrates stored in cells. However, it is not known whether TFEB plays a role in cholesterol clearance in NPC disease. Here, we show that transgenic overexpression of TFEB, but not TFE3 (another member of MiTF family) facilitates cholesterol clearance in various NPC1 cell models. Pharmacological activation of TFEB by sulforaphane (SFN), a previously identified natural small-molecule TFEB agonist by us, can dramatically ameliorate cholesterol accumulation in human and mouse NPC1 cell models. In NPC1 cells, SFN induces TFEB nuclear translocation via a ROS-Ca2+-calcineurin dependent but MTOR-independent pathway and upregulates the expression of TFEB-downstream genes, promoting lysosomal exocytosis and biogenesis. While genetic inhibition of TFEB abolishes the cholesterol clearance and exocytosis effect by SFN. In the NPC1 mouse model, SFN dephosphorylates/activates TFEB in brain and exhibits potent efficacy of rescuing the loss of Purkinje cells and body weight. Hence, pharmacological upregulating lysosome machinery via targeting TFEB represents a promising approach to treat NPC and related lysosomal storage diseases, and provides the possibility of TFEB agonists ie SFN as potential NPC therapeutic candidates.

molecular biology↗

bioRxiv2021

ASFV pD345L protein negatively regulates NF-kB signaling through inhibiting IKK kinase activity

NF-{kappa}B is a critical transcription factor in immediate early viral infection, including African swine fever virus (ASFV), playing an important role in inflammation response and expression of antiviral genes. ASFV encodes for more than 151 proteins by its own transcription machinery and possesses a great capacity to evade or subvert antiviral innate immune responses. A couple of such viral proteins have been reported, but many remain unknown. Here, we showed that pD345L, an ASFV-encoded lambda-like exonuclease, is an inhibitor of cGAS/STING mediated NF-{kappa}B signaling by blocking IKK/{beta} kinase activity. Specifically, we showed that overexpression of pD345L suppresses cGAS/STING induced IFN{beta} and NF-{kappa}B activation, resulting in decreased transcription of IFN{beta} and several pro-inflammatory cytokines, including IL-1, IL-6, IL-8, and TNF. In addition, we showed that pD345L targeted at or downstream of IKK and upstream of p65. Importantly, we found that pD345L associates with KD and HLH domains of IKK and LZ domain of IKK{beta}, and thus interrupts their kinase activity on downstream substrate I{kappa}B. Finally, we showed that pD345L inhibition of NF-{kappa}B signaling was independent of its exonuclease activity. Taken together, we concluded that pD345L blocks IKK /{beta} kinase activity by protein-protein interaction and thus disrupts cGAS/STING mediated NF-{kappa}B signaling. ImportanceAfrican Swine Fever (ASF) is one of the most devastating and economically significant swine diseases caused by African swine fever virus (ASFV). Since expanding of ASFV affected areas into Asian countries, especially China, an effective vaccine is urgently needed more than ever. Therefore, it is of great significance to understand the interaction between ASFV infection and host immune responses. The NF-{kappa}B signaling plays a central role in innate and acquired immune responses. Activation of I{kappa}B kinase (IKK) complex is a key step of both canonical and non-canonical NF-{kappa}B pathways, and is commonly targeted by different viruses. But no ASFV protein has been shown to regulate IKK yet. In this study, we demonstrated that pD345L blocks IKK/{beta} kinase activity by protein-protein interaction and thus disrupts cGAS/STING mediated NF-{kappa}B signaling. It has been shown that conventional vaccine development approaches are proven to be inapplicable to ASFV. Neither subunit nor DNA vaccine provides efficient protection. Gene deleted live-attenuated vaccine candidates render adequate protection, but establishment of chronic or persistent infection in vaccinated animals and risk of recombination with filed strains are key challenges. To overcome these, one potential strategy would be generation of replication-defective viruses. As a lambda-like exonuclease, pD345L plays a critical role in ASFV replication and ASFV deficient in D345L cannot be rescued. Given the dual role of pD345L in virus replication and immune evasion, it may serve as a potential target for replication-defective virus vaccine development.

microbiology↗