AUTACs: Cargo-Specific Degraders Using Selective Autophagy.

Protein silencing represents an essential tool in biomedical research. Targeted protein degradation (TPD) strategies exemplified by PROTACs are rapidly emerging as modalities in drug discovery. However, the scope of current TPD techniques is limited because many intracellular materials are not substrates of proteasomal clearance. Here, we described a novel targeted-clearance strategy (autophagy-targeting chimera [AUTAC]) that contains a degradation tag (guanine derivatives) and a warhead to provide target specificity. As expected from the substrate scope of autophagy, AUTAC degraded fragmented mitochondria as well as proteins. Mitochondria-targeted AUTAC accelerated both the removal of dysfunctional fragmented mitochondria and the biogenesis of functionally normal mitochondria in patient-derived fibroblast cells. Cytoprotective effects against acute mitochondrial injuries were also seen. Canonical autophagy is viewed as a nonselective bulk decomposition system, and none of the available autophagy-inducing agents exhibit useful cargo selectivity. With its target specificity, AUTAC provides a new modality for research on autophagy-based drugs.

8-Nitro-cGMP attenuates context-dependent fear memory in mice.

We previously reported that 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is endogenously produced via nitric oxide/reactive oxygen species signaling pathways and it reacts with protein thiol residues to add cGMP structure to proteins through S-guanylation. S-Guanylation occurs on synaptosomal-associated protein 25 (SNAP-25), which is a part of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that regulates exocytosis. However, the biological relevance of 8-nitro-cGMP in the nervous system remains unclear. Here, we investigated the effects of intracerebroventricular (icv) infusion of 8-nitro-cGMP on mouse brain functions. The results of an open-field test and fear-conditioning task revealed that icv infusion of 8-nitro-cGMP decreased the vertical activity and context-dependent fear memory of mice, which are both associated with the hippocampus. Immunohistochemical analysis revealed increased c-Fos-positive cells in the dentate gyrus in 8-nitro-cGMP-infused mice. Further, biochemical analyses showed that icv infusion of 8-nitro-cGMP increased S-guanylated proteins including SNAP-25 and SNARE complex formation as well as decreased complexes containing complexin, which regulates exocytosis by binding to the SNARE complex, in the hippocampus. These findings suggest that accumulation of 8-nitro-cGMP in the hippocampus affects its functions, including memory, via S-guanylation of hippocampal proteins such as SNAP-25.

Modification of Tau by 8-Nitroguanosine 3',5'-Cyclic Monophosphate (8-Nitro-cGMP): EFFECTS OF NITRIC OXIDE-LINKED CHEMICAL MODIFICATION ON TAU AGGREGATION.

Neurofibrillar tangles caused by intracellular hyperphosphorylated tau inclusion and extracellular amyloid ß peptide deposition are hallmarks of Alzheimer's disease. Tau contains one or two cysteine residues in three or four repeats of the microtubule binding region following alternative splicing of exon 10, and formation of intermolecular cysteine disulfide bonds accelerates tau aggregation. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) acts as a novel second messenger of nitric oxide (NO) by covalently binding cGMP to cysteine residues by electrophilic properties, a process termed protein S-guanylation. Here we studied S-guanylation of tau and its effects on tau aggregation. 8-Nitro-cGMP exposure induced S-guanylation of tau both in vitro and in tau-overexpressed HEK293T cells. S-guanylated tau inhibited heparin-induced tau aggregation in a thioflavin T assay. Atomic force microscopy observations indicated that S-guanylated tau could not form tau granules and fibrils. Further biochemical analyses showed that S-guanylated tau was inhibited at the step of tau oligomer formation. In P301L tau-expressing Neuro2A cells, 8-nitro-cGMP treatment significantly reduced the amount of sarcosyl-insoluble tau. NO-linked chemical modification on cysteine residues of tau could block tau aggregation, and therefore, increasing 8-nitro-cGMP levels in the brain could become a potential therapeutic strategy for Alzheimer's disease.

8-Nitro-cGMP: A Novel Protein-Reactive cNMP and Its Emerging Roles in Autophagy.

Nitric oxide (NO) raises the intracellular 3',5'-cyclic guanosine monophosphate (cGMP) level through the activation of soluble guanylate cyclase and, in the presence of reactive oxygen species (ROS), reacts with biomolecules to produce nitrated cGMP derivatives. 8-Nitro-cGMP was the first endogenous cGMP derivative discovered in mammalian cells (2007) and was later found in plant cells. Among the six nitrogen atoms in this molecule, the one in the nitro group (NO2) comes from NO. This chapter asserts that this newly found cGMP is undoubtedly one of the major physiological cNMPs. Multiple studies suggest that its intracellular abundance might exceed that of unmodified cGMP. The characteristic chemical feature of 8-nitro-cGMP is its ability to modify proteinous cysteine residues via a stable sulfide bond. In this posttranslational modification, the nitro group is detached from the guanine base. This modification, termed "protein S-guanylation," is known to regulate the physiological functions of several important proteins. Furthermore, 8-nitro-cGMP participates in the regulation of autophagy. For example, in antibacterial autophagy (xenophagy), S-guanylation accumulates around invading bacterial cells and functions as a "tag" for subsequent clearance of the organism via ubiquitin modifications. This finding suggests the existence of a system for recognizing the cGMP structure on proteins. Autophagy induction by 8-nitro-cGMP is mechanistically distinct from the well-described starvation-induced autophagy and is independent of the action of mTOR, the master regulator of canonical autophagy.

Endogenous occurrence of protein S-guanylation in Escherichia coli: Target identification and genetic regulation.

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E. coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E. coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E. coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E. coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5'-triphosphate. More importantly, E. coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E. coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections.

Formation, signaling functions, and metabolisms of nitrated cyclic nucleotide.

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a unique derivative of guanosine 3',5'-cyclic monophosphate (cGMP) formed in mammalian and plant cells in response to production of nitric oxide and reactive oxygen species. 8-Nitro-cGMP possesses signaling activity inherited from parental cGMP, including induction of vasorelaxation through activation of cGMP-dependent protein kinase. On the other hand, 8-nitro-cGMP mediates cellular signaling that is not observed for native cGMP, e.g., it behaves as an electrophile and reacts with protein sulfhydryls, which results in cGMP adduction to protein sulfhydryls (protein S-guanylation). Several proteins have been identified as targets for endogenous protein S-guanylation, including Kelch-like ECH-associated protein 1 (Keap1), H-Ras, and mitochondrial heat shock proteins. 8-Nitro-cGMP signaling via protein S-guanylation of those proteins may have evolved to convey adaptive cellular stress responses. 8-Nitro-cGMP may not undergo conventional cGMP metabolism because of its resistance to phosphodiesterases. Hydrogen sulfide has recently been identified as a potent regulator for metabolisms of electrophiles including 8-nitro-cGMP, through sulfhydration of electrophiles, e.g., leading to the formation of 8-SH-cGMP. Better understanding of the molecular basis for the formation, signaling functions, and metabolisms of 8-nitro-cGMP would be useful for the development of new diagnostic approaches and treatment of diseases related to oxidative stress and redox metabolisms.

Selective autophagy as the basis of autophagy-based degraders.

Degrader technologies, which enable the chemical knockdown of disease-causing proteins, are promising for drug discovery. After two decades of research, degraders using the ubiquitin-proteasome system (UPS) are currently in clinical trials. However, the UPS substrates are mainly limited to soluble proteins. Autophagy-targeting chimeras and autophagosome-tethering compounds are degraders that use autophagy, which has functions complementary to the UPS. They can degrade organelles and aggregate-prone proteins, making them promising treatments against age-related conditions such as mitochondrial dysfunction and neurodegenerative diseases. The molecular mechanism of selective autophagy is an ongoing research topic, which explains why autophagy-based degraders were not available until recently. In this review, we introduce four classifications of selective autophagy mechanisms to facilitate the understanding of the degrader design.

Nitrated Nucleotides: New Players in Signaling Pathways of Reactive Nitrogen and Oxygen Species in Plants.

Nitration of diverse biomolecules, including proteins, lipids and nucleic acid, by reactive nitrogen species represents one of the key mechanisms mediating nitric oxide (NO) biological activity across all types of organisms. 8-nitroguanosine 3'5'-cyclic monophosphate (8-nitro-cGMP) has been described as a unique electrophilic intermediate involved in intracellular redox signaling. In animal cells, 8-nitro-cGMP is formed from guanosine-5'-triphosphate by a combined action of reactive nitrogen (RNS) and oxygen species (ROS) and guanylate cyclase. As demonstrated originally in animal models, 8-nitro-cGMP shows certain biological activities closely resembling its analog cGMP; however, its regulatory functions are mediated mainly by its electrophilic properties and chemical interactions with protein thiols resulting in a novel protein post-translational modification termed S-guanylation. In Arabidopsis thaliana, 8-nitro-cGMP was reported to mediate NO-dependent signaling pathways controlling abscisic acid (ABA)-induced stomatal closure, however, its derivative 8-mercapto-cGMP (8-SH-cGMP) was later shown as the active component of hydrogen sulfide (H2S)-mediated guard cell signaling. Here we present a survey of current knowledge on biosynthesis, metabolism and biological activities of nitrated nucleotides with special attention to described and proposed functions of 8-nitro-cGMP and its metabolites in plant physiology and stress responses.

8-Nitro-cGMP Attenuates the Interaction between SNARE Complex and Complexin through S-Guanylation of SNAP-25.

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is the second messenger in nitric oxide/reactive oxygen species redox signaling. This molecule covalently binds to protein thiol groups, called S-guanylation, and exerts various biological functions. Recently, we have identified synaptosomal-associated protein 25 (SNAP-25) as a target of S-guanylation, and demonstrated that S-guanylation of SNAP25 enhanced SNARE complex formation. In this study, we have examined the effects of S-guanylation of SNAP-25 on the interaction between the SNARE complex and complexin (cplx), which binds to the SNARE complex with a high affinity. Pull-down assays and coimmunoprecipitation experiments have revealed that S-guanylation of Cys90 in SNAP-25 attenuates the interaction between the SNARE complex and cplx. In addition, blue native-PAGE followed by Western blot analysis revealed that the amount of cplx detected at a high molecular weight decreased upon 8-nitro-cGMP treatment in SH-SY5Y cells. These results demonstrated for the first time that S-guanylation of SNAP-25 attenuates the interaction between the SNARE complex and cplx.

8-Nitro-cGMP Enhances SNARE Complex Formation through S-Guanylation of Cys90 in SNAP25.

Nitrated guanine nucleotide 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP formation and its protein targets in the normal brain have not been identified. Here, we investigated 8-nitro-cGMP generation and protein S-guanylation in the rodent brain. Immunohistochemistry indicated that 8-nitro-cGMP was produced by neurons, such as pyramidal cells and interneurons. Using liquid chromatography-tandem mass spectrometry, we determined endogenous 8-nitro-cGMP levels in the brain as 2.92 ± 0.10 pmol/mg protein. Based on S-guanylation proteomics, we identified several S-guanylated neuronal proteins, including SNAP25 which is a core member of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. SNAP25 post-translational modification including palmitoylation, phosphorylation, and oxidation, are known to regulate neurotransmission. Our results demonstrate that S-guanylation of SNAP25 enhanced the stability of the SNARE complex, which was further promoted by Ca(2+)-dependent activation of neuronal nitric oxide synthase. Using site-directed mutagenesis, we identified SNAP25 cysteine 90 as the main target of S-guanylation which enhanced the stability of the SNARE complex. The present study revealed a novel target of redox signaling via protein S-guanylation in the nervous system and provided the first substantial evidence of 8-nitro-cGMP function in the nervous system.

A new paradigm for antimicrobial host defense mediated by a nitrated cyclic nucleotide.

Nitric oxide (NO), produced by inducible NO synthase (iNOS) during infection, plays a crucial role in host defense mechanisms. Salmonella typhimurium infection in mice is associated with excessive production of NO from iNOS as a host defense response. An important cytoprotective and antimicrobial function of NO is mediated by induction of heme oxygenase (HO)-1. The signaling mechanism of NO-dependent HO-1 induction has remained unclear, however. We recently discovered a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), which is formed via guanine nitration with NO and reactive oxygen species. iNOS-dependent 8-nitro-cGMP formation and HO-1 induction were identified in Salmonella-infected mice. Extensive apoptosis observed with iNOS-deficient macrophages infected with Salmonella was remarkably suppressed via HO-1 induced by 8-nitro-cGMP formed in cells. This cytoprotective signaling appears to be mediated by the reaction of 8-nitro-cGMP with protein sulfhydryls to generate a novel post-translational modification named protein S-guanylation. We also found that 8-nitro-cGMP specifically S-guanylates Keap1, a negative regulator of transcription factor Nrf2, which in turn up-regulates transcription of HO-1. Here, we discuss the unique mechanism of NO-mediated host defense that operates via formation of a novel signaling molecule - 8-nitro-cGMP - during microbial infections.