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Toll-like receptors (TLRs) play a crucial role in several innate immune responses by regulating autophagy, but little is known about how TLR signaling controls autophagy. Here we demonstrate that p62/ SQSTM1 is required for TLR4-mediated autophagy, which we show as selective autophagy of aggresome-like induced structures (ALIS). Treatment with LPS or Escherichia coli induced LC3 + dot-like structures, and their assembly, but not lysosomal degradation, occurred independently of classic autophagic machinery. Microscopic and ultrastructural analyses showed that p62 is a component of the induced LC3+ dots and these TLR4-induced p62 + structures resemble ALIS. The levels of p62 mRNA and protein were increased in TLR4-activated cells and knockdown of p62 suppressed the ALIS formation and LC3-II conversion. The accumulation of p62 and ALIS required activation of Nrf2 by reactive oxygen species-p38 axis-dependent TLR4/MyD88 signaling, suggesting a link between innate immune and oxidative-stress responses. These findings indicate that TLR4-driven induction of p62 plays an essential role in the formation and the autophagic degradation of ALIS, which might be critical for regulating host defense.T oll-like receptors (TLRs) are pattern recognition receptors that have evolved to detect infection by recognizing the conserved pathogen-associated molecular patterns and trigger innate immune responses to defend against invading microorganisms (1, 2). Engagement of TLR4 with Gram-negative bacteria, such as Escherichia coli, via bacterial membrane component LPS, triggers the assembly of Toll/IL-1R (TIR) domain-containing adaptors, such as MyD88 (myeloid differentiation primary response gene 88) and TRIF (TIR domain-containing adaptor inducing IFN-β). MyD88-dependent TLR4 signaling ignites activation of cellular signaling molecules, IRAK4 (IL-1R-associated kinase 4) and TRAF6 (TNFR-associated factor 6), the downstream kinases, IKK, p38, and JNK, leading to the induction of inflammatory cytokines, including IL-1β and IL-6. In contrast, TRIF-mediated pathways mainly regulate antiviral type I IFN responses through TRAF3-TBK1-IKKi and the activation of a transcription factor IFN-regulating factor 3. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional factor that is responsible for cellular defense against oxidative stress also contributes to TLR4-mediated innate immune responses (3).Recent research has implicated evolutionarily conserved macroautophagy (hereafter referred to as autophagy) in innate immune responses (4, 5). Autophagy is a bulk degradation system by which cytoplasmic materials are engulfed into double-membrane vesicles, known as autophagosomes, and delivered to lysosomes for degradation (6, 7). In the past decade, the basic steps involved in autophagy have been elucidated. Autophagy-related gene products, such as ATG5 and ATG7, mediate the conjugation of phosphatidylethanolamine (PE) to microtubule-associated protein 1 light-chain 3 (LC3), an essential step in autophagy, by mechanisms similar to ubiquit...

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1′, which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.

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We report a simple and readily available fluorescent probe for rapid, specific, and ratiometric fluorescent detection of the biologically important cysteine (Cys). This probe uses a visible-light excitable excited-state intramolecular proton transfer (ESIPT) dye (4'-dimethylamino-3-hydroxyflavone) as the fluorophore and an acrylate group as the ESIPT blocking agent as well as the recognition unit. Cleavage of the acrylate moiety can be achieved specifically and rapidly by Cys in aqueous solution under mild conditions, which leads to restore the ESIPT process and enables the probe to show a rapid, ratiometric fluorescent detection process for Cys with high selectivity over various analytes, including homocysteine (Hcy) and glutathione (GSH). The detection limit of this probe for Cys was found to be ∼0.2 μM and bioimaging of intracellular Cys by this probe was successfully applied in living cells, indicating that this probe holds great potential for biological applications.

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