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Ubiquitination and sumoylation are two important posttranslational modifications in cells. RING (Really Interesting New Gene)-type E3 ligases play essential roles in regulating a plethora of biological processes such as cell survival and death. In our previous study, we performed a microarray using inputs from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine and identified a novel RING-type E3 ligase, RNF166. We showed that RNF166 exerts proapoptotic effects via ubiquitin-dependent degradation of X-linked inhibitor of apoptosis and subsequent overactivation of caspase-dependent neuronal death following 6-hydroxydopamine treatment. In the present study, we further expanded the list of RNF166's binding substrates using mass spectral analyses of immunoprecipitates obtained from RNF166-overexpressing HEK293 cells. Poly (ADP-ribose) polymerase 1, ATPase WRNIP1, X-ray repair cross-complementing protein 5 (Ku80), and replication protein A 70 were identified as potential binding partners of RNF166. Additionally, we confirmed that RNF166 interacts with and forms lysine 63-linked polyubiquitin chains in Ku80. Consequently, these events promoted the increased stability of Ku80. Intriguingly, we found that RNF166 also contains distinct consensus sequences termed SUMO-interacting motifs and interacts with apoptosis signal-regulating kinase 1 (ASK1). We determined that RNF166 induces the sumoylation of ASK1. Overall, our data provide novel evidence that RNF166 has a dual function of Lys63-linked ubiquitination and sumoylation of its cellular targets.

Protein S-nitros(yl)ation (SNO) is a posttranslational modification involved in diverse processes in health and disease and can contribute to synaptic damage in Alzheimer’s disease (AD). To identify SNO proteins in AD brains, we used triaryl phosphine ( SNO TRAP) combined with mass spectrometry (MS). We detected 1449 SNO proteins with 2809 SNO sites, representing a wide range of S-nitrosylated proteins in 40 postmortem AD and non-AD human brains from patients of both sexes. Integrative protein ranking revealed the top 10 increased SNO proteins, including complement component 3 (C3), p62 (SQSTM1), and phospholipase D3. Increased levels of S-nitrosylated C3 were present in female over male AD brains. Mechanistically, we show that formation of SNO-C3 is dependent on falling β-estradiol levels, leading to increased synaptic phagocytosis and thus synapse loss and consequent cognitive decline. Collectively, we demonstrate robust alterations in the S-nitrosoproteome that contribute to AD pathogenesis in a sex-dependent manner.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

Rosemary (Rosmarinus officinalis [family Lamiaceae]), an herb of economic and gustatory repute, is employed in traditional medicines in many countries. Rosemary contains carnosic acid (CA) and carnosol (CS), abietane-type phenolic diterpenes, which account for most of its biological and pharmacological actions, although claims have also been made for contributions of another constituent, rosmarinic acid. This review focuses on the potential applications of CA and CS for Alzheimer’s disease (AD), Parkinson’s disease (PD), and coronavirus disease 2019 (COVID-19), in part via inhibition of the NLRP3 inflammasome. CA exerts antioxidant, anti-inflammatory, and neuroprotective effects via phase 2 enzyme induction initiated by activation of the KEAP1/NRF2 transcriptional pathway, which in turn attenuates NLRP3 activation. In addition, we propose that CA-related compounds may serve as therapeutics against the brain-related after-effects of SARS-CoV-2 infection, termed “long-COVID.” One factor that contributes to COVID-19 is cytokine storm emanating from macrophages as a result of unregulated inflammation in and around lung epithelial and endovascular cells. Additionally, neurological aftereffects such as anxiety and “brain fog” are becoming a major issue for both the pandemic and post-pandemic period. Many reports hold that unregulated NLRP3 inflammasome activation may potentially contribute to the severity of COVID-19 and its aftermath. It is therefore possible that suppression of NLRP3 inflammasome activity may prove efficacious against both acute lung disease and chronic neurological after-effects. Because CA has been shown to not only act systemically but also to penetrate the blood–brain barrier and reach the brain parenchyma to exert neuroprotective effects, we discuss the evidence that CA or rosemary extracts containing CA may represent an effective countermeasure against both acute and chronic pathological events initiated by SARS-CoV-2 infection as well as other chronic neurodegenerative diseases including AD and PD.

Reactive nitrogen species (RNS) such as nitric oxide (NO) act as signaling molecules that mediate both physiological and pathological processes. Recent evidence in biological systems has shown the ubiquitous nature of a redox-mediated post-translational modification of specific cysteine thiols (or more properly, thiolate anion S-) by NO, most likely in the form of a nitrosonium cation intermediate (NO+). This nitrosation reaction is now termed protein S-nitrosylation and represents a key mechanism for the biological actions of NO. Like other post-translational modifications, S-nitrosylation can affect ion channel activity, enzymatic function, protein conformation, protein-protein interactions, and protein trafficking and localization. In the nervous system, S-nitrosylation was first discovered on the NMDA type of glutamate receptor (NMDAR) and occurs on many proteins affecting neuronal survival, synaptic function, neurogenesis, and other aspects of neuronal development. This chapter provides an overview of the molecular and cellular mechanisms by which RNS regulate neuronal differentiation via S-nitrosylation of proteins such as histone deacetylation enzyme HDAC2 and transcription factor MEF2. Dysregulation of the S-nitrosylation signaling network can impair normal brain development/neurogenesis as well as adult neurogenesis, contributing to neurodevelopmental and neurodegenerative conditions. Further investigation into the mechanism of S-nitrosylation-regulated neuronal differentiation should elucidate pathogenic features of these disorders.

Ubiquitination and sumoylation are two important posttranslational modifications in cells. RING (Really Interesting New Gene)-type E3 ligases play essential roles in regulating a plethora of biological processes such as cell survival and death. In our previous study, we performed a microarray using inputs from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine and identified a novel RING-type E3 ligase, RNF166. We showed that RNF166 exerts proapoptotic effects via ubiquitin-dependent degradation of X-linked inhibitor of apoptosis and subsequent overactivation of caspase-dependent neuronal death following 6-hydroxydopamine treatment. In the present study, we further expanded the list of RNF166's binding substrates using mass spectral analyses of immunoprecipitates obtained from RNF166-overexpressing HEK293 cells. Poly (ADP-ribose) polymerase 1, ATPase WRNIP1, X-ray repair cross-complementing protein 5 (Ku80), and replication protein A 70 were identified as potential binding partners of RNF166. Additionally, we confirmed that RNF166 interacts with and forms lysine 63-linked polyubiquitin chains in Ku80. Consequently, these events promoted the increased stability of Ku80. Intriguingly, we found that RNF166 also contains distinct consensus sequences termed SUMO-interacting motifs and interacts with apoptosis signal-regulating kinase 1 (ASK1). We determined that RNF166 induces the sumoylation of ASK1. Overall, our data provide novel evidence that RNF166 has a dual function of Lys63-linked ubiquitination and sumoylation of its cellular targets.

Protein S-nitrosylation is known to regulate enzymatic function. Here, we report that nitric oxide (NO)-related species can contribute to Alzheimer's disease (AD) by S-nitrosylating the lysosomal protease cathepsin B (forming SNO-CTSB), thereby inhibiting CTSB activity. This posttranslational modification inhibited autophagic flux, increased autolysosomal vesicles, and led to accumulation of protein aggregates. CA-074Me, a CTSB chemical inhibitor, also inhibited autophagic flux and resulted in accumulation of protein aggregates similar to the effect of SNO-CTSB. Inhibition of CTSB activity also induced caspase-dependent neuronal apoptosis in mouse cerebrocortical cultures. To examine which cysteine residue(s) in CTSB are S-nitrosylated, we mutated candidate cysteines and found that three cysteines were susceptible to S-nitrosylation. Finally, we observed an increase in SNO-CTSB in both 5XFAD transgenic mouse and flash-frozen postmortem human AD brains. These results suggest that S-nitrosylation of CTSB inhibits enzymatic activity, blocks autophagic flux, and thus contributes to AD pathogenesis.

Significance: Physiological concentrations of nitric oxide (NO(•)) and related reactive nitrogen species (RNS) mediate multiple signaling pathways in the nervous system. During inflammaging (chronic low-grade inflammation associated with aging) and in neurodegenerative diseases, excessive RNS contribute to synaptic and neuronal loss. “NO signaling” in both health and disease is largely mediated through protein S-nitrosylation (SNO), a redox-based posttranslational modification with “NO” (possibly in the form of nitrosonium cation [NO(+)]) reacting with cysteine thiol (or, more properly, thiolate anion [R-S(−)]). Recent Advances: Emerging evidence suggests that S-nitrosylation occurs predominantly via transnitros(yl)ation. Mechanistically, the reaction involves thiolate anion, as a nucleophile, performing a reversible nucleophilic attack on a nitroso nitrogen to form an SNO-protein adduct. Prior studies identified transnitrosylation reactions between glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-nuclear proteins, thioredoxin-caspase-3, and X-linked inhibitor of apoptosis (XIAP)-caspase-3. Recently, we discovered that enzymes previously thought to act in completely disparate biochemical pathways can transnitrosylate one another during inflammaging in an unexpected manner to mediate neurodegeneration. Accordingly, we reported a concerted tricomponent transnitrosylation network from Uch-L1-to-Cdk5-to-Drp1 that mediates synaptic damage in Alzheimer's disease. Critical Issues: Transnitrosylation represents a critical chemical mechanism for transduction of redox-mediated events to distinct subsets of proteins. Although thousands of thiol-containing proteins undergo S-nitrosylation, how transnitrosylation regulates a myriad of neuronal attributes is just now being uncovered. In this review, we highlight recent progress in the study of the chemical biology of transnitrosylation between proteins as a mechanism of disease. Future Directions: We discuss future areas of study of protein transnitrosylation that link our understanding of aging, inflammation, and neurodegenerative diseases. Antioxid. Redox Signal. 35, 531–550.

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of “sporadic” cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.

A cascade of NO in Alzheimer's disease One of the ill effects of the amyloid-β peptide that accumulates in Alzheimer's disease (AD) is the promotion of the production of nitric oxide (NO) and consequent nitrosylation of thiols in proteins such as dynamin-related protein 1 (Drp1), which can lead to loss of neuronal synapses. Nakamura et al. found that this S-nitrosylation occurs in an unusual way. They detected a series of transnitrosylation events in which an NO group is passed between at least three proteins. The deubiquinating enzyme Uch-L1 was S-nitrosylated in brains from human AD patients or in mouse models of AD. Uch-L1 could lead to S-nitrosylation of Drp1 after transferring the NO group to another enzyme, Cdk5 (cyclin-dependent kinase 5). The results implicate a mechanism in which unrelated enzymes might aberrantly function together to disrupt brain function. Science , this issue p. eaaw0843

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by heterozygous mutations in the TSC1 or TSC2 gene. TSC is often associated with neurological, cognitive, and behavioral deficits. TSC patients also express co-morbidity with anxiety and mood disorders. The mechanism of pathogenesis in TSC is not entirely clear, but TSC-related neurological symptoms are accompanied by excessive glutamatergic activity and altered synaptic spine structures. To address whether extrasynaptic (e)NMDA-type glutamate receptor (NMDAR) antagonists, as opposed to antagonists that block physiological phasic synaptic activity, can ameliorate the synaptic and behavioral features of this disease, we utilized the Tsc2+/− mouse model of TSC to measure biochemical, electrophysiological, histological, and behavioral parameters in the mice. We found that antagonists that preferentially block tonic activity as found at eNMDARs, particularly the newer drug NitroSynapsin, provide biological and statistically significant improvement in Tsc2+/− phenotypes. Accompanying this improvement was correction of activity in the p38 MAPK-TSC-Rheb-mTORC1-S6K1 pathway. Deficits in hippocampal long-term potentiation (LTP), histological loss of synapses, and behavioral fear conditioning in Tsc2+/− mice were all improved after treatment with NitroSynapsin. Taken together, these results suggest that amelioration of excessive excitation, by limiting aberrant eNMDAR activity, may represent a novel treatment approach for TSC.

Mutations in PARK6 (PINK1) and PARK2 (Parkin) are linked to rare familial cases of Parkinson's disease (PD). Mutations in these genes result in pathological dysregulation of mitophagy, contributing to neurodegeneration. Here, we report that environmental factors causing a specific posttranslational modification on PINK1 can mimic these genetic mutations. We describe a molecular mechanism for impairment of mitophagy via formation of S-nitrosylated PINK1 (SNO-PINK1). Mitochondrial insults simulating age- or environmental-related stress lead to increased SNO-PINK1, inhibiting its kinase activity. SNO-PINK1 decreases Parkin translocation to mitochondrial membranes, disrupting mitophagy in cell lines and human-iPSC-derived neurons. We find levels of SNO-PINK1 in brains of α-synuclein transgenic PD mice similar to those in cell-based models, indicating the pathophysiological relevance of our findings. Importantly, SNO-PINK1-mediated deficits in mitophagy contribute to neuronal cell death. These results reveal a direct molecular link between nitrosative stress, SNO-PINK1 formation, and mitophagic dysfunction that contributes to the pathogenesis of PD. Nitrosative stress and mitochondrial dysfunction represent key pathological events in Parkinson's disease. Oh et al. identify a molecular link between these events in which increased nitric oxide (NO)-related species S-nitrosylate a critical thiol group in PINK1, thus compromising its ability to eliminate damaged mitochondria via mitophagy., National Institutes of Health (U.S.) (Grant R01 NS086890), National Institutes of Health (U.S.) (Grant P01 ES016738), National Institutes of Health (U.S.) (Grant DP1 DA041722), National Institutes of Health (U.S.) (Grant RF1 AG057409), National Institutes of Health (U.S.) (Grant R01 AG056259)

Bax is translocated into the mitochondrial membrane and oligomerized therein to initiate mitochondrial apoptotic signaling. Our previous study indicated that reactive oxygen species (ROS)-mediated activation of mitogen-activated protein kinase (MAPK) and caspase is critically involved in 6-hydroxydopamine (6-OHDA)-mediated neurodegeneration. Here, we specifically attempted to examine whether and how these death signaling pathways may be linked to Bax translocation and oligomerization. We found that 6-OHDA treatment triggered translocation and oligomerization of Bax onto the mitochondria in MN9D dopaminergic neuronal cells. These events preceded cytochrome c release into the cytosol. Cross-linking assay revealed that co-treatment with a ROS scavenger or a pan-caspase inhibitor inhibited 6-OHDA-induced Bax oligomerization. Among several candidates of ROS-activated MAPKs and caspases, we found that co-treatment with PD169316 or VDVAD specifically inhibited 6-OHDA-induced Bax oligomerization, suggesting critical involvement of p38 MAPK and caspase-2. Consequently, overexpression of a dominant negative form of p38 MAPK or a shRNA-mediated knockdown of caspase-2 indeed inhibited 6-OHDA-induced Bax oligomerization. However, activation of p38 MAPK and caspase-2 was independently linked to oligomerization of Bax. This specificity was largely confirmed with a Bax 6A7 antibody known to detect activated forms of Bax on the mitochondria. Taken together, our data suggest that there is an independent amplification loop of Bax translocation and oligomerization via caspase-2 and p38 MAPK during ROS-mediated dopaminergic neurodegeneration.