Sharadha Dayalan Naidu

Transcription factors Hsf1 and Nrf2 engage in crosstalk for cytoprotection

Transcription factors heat shock factor (Hsf)1 and nuclear factor-erythroid 2 p45-related factor (Nrf)2 are critical for adaptation and survival. Each is maintained at low basal levels, but is robustly activated by various stimuli, including cysteine-reactive small molecules (inducers). Although each is regulated by distinct mechanisms, it is emerging that these transcription factors engage in crosstalk by sharing overlapping transcriptional targets, such as heat shock protein (HSP)70, p62, and activating transcription factor (ATF)3, and in certain cases, compensating for each other. Critically, activation of Hsf1 or Nrf2 affects the cellular redox balance by promoting the reduced state. Conversely, deletion of Hsf1 or Nrf2 is associated with oxidative stress and impaired mitochondrial function. Transient activation of Hsf1 and Nrf2 is cytoprotective, but their persistent upregulation may be detrimental, causing cardiomyopathy or accelerating carcinogenesis, and should be considered when designing strategies for disease prevention and treatment.

Regulation of the mammalian heat shock factor 1

Living organisms are endowed with the capability to tackle various forms of cellular stress due to the presence of molecular chaperone machinery complexes that are ubiquitous throughout the cell. During conditions of proteotoxic stress, the transcription factor heat shock factor 1 (HSF1) mediates the elevation of heat shock proteins, which are crucial components of the chaperone complex machinery and function to ameliorate protein misfolding and aggregation and restore protein homeostasis. In addition, HSF1 orchestrates a versatile transcriptional programme that includes genes involved in repair and clearance of damaged macromolecules and maintenance of cell structure and metabolism, and provides protection against a broad range of cellular stress mediators, beyond heat shock. Here, we discuss the structure and function of the mammalian HSF1 and its regulation by post‐translational modifications (phosphorylation, sumoylation and acetylation), proteasomal degradation, and small‐molecule activators and inhibitors.

Dopamine transporter changes in neuropsychiatric disorders.

Publisher Summary The highest concentrations of dopamine transporters (DATs) are found in the basal ganglia, corresponding to the amount of dopamine (DA) nerve terminals in this brain region. Numerous studies demonstrating parallel losses of DA levels and DAT after lesions of nigrostriatal DA neurons suggest that the density of DAT is an excellent marker of the structural integrity of the dopaminergic system. In vivo imaging of DAT has been performed mainly in patients with Parkinsons disease to demonstrate the marked loss of nigrostriatal DA nerve terminals in this disorder. However, positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging of DAT have been shown to be useful in various other neurodegenerative disorders, including Lesch-Nyhan disease (LND), Rett syndrome, Tourettes syndrome, stimulant abuse, and progressive supranuclear palsy. A marked reduction of striatal DAT densities has been reported in methamphetamine treated animals. In baboon studies with methamphetamine treatment, a dose-related reduction in DAT densities occurs. These studies are followed by postmortem analysis of neurochemical parameters. In recent years, the synthetic amphetamine analogue methcathinone (2-methylamino-1-phenylpropanone, ephedrone, “Cat”) has emerged as a recreational drug of abuse. In animals, methcathinone, like methamphetamine, produces toxic effects on brain DA and serotonin neurons. Moreover, studies in human subjects with a history of methcathinone abuse suggest a DAT decline as well.

Activation of Nrf2 Signaling Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.

Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

ABSTRACT Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients

Significance Chronic neuroinflammation and oxidative stress are likely complicit in driving disease progression in Huntingtons disease (HD). Here, we describe the mechanism of action of a unique chemical scaffold that is highly selective for activation of NRF2, the master transcriptional regulator of cellular antiinflammatory and antioxidant defense genes. The use of this scaffold revealed that NRF2 activation responses were muted in HD patient-derived neural stem cells, suggesting increased susceptibility of this critical renewable cell population to oxidative stress in HD brain. However, pharmacological activation of NRF2 was able to repress inflammatory responses in mouse microglia and astrocytes, the principal cellular mediators of neuroinflammation, and in blood monocytes from HD patients. Our results suggest multiple protective benefits of NRF2 activation for HD patients. The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntingtons disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

Transcription factors NRF2 and HSF1 have opposing functions in autophagy

Autophagy plays a critical role in the maintenance of cellular homeostasis by degrading proteins, lipids and organelles. Autophagy is activated in response to stress, but its regulation in the context of other stress response pathways, such as those mediated by heat shock factor 1 (HSF1) and nuclear factor-erythroid 2 p45-related factor 2 (NRF2), is not well understood. We found that the Michael acceptor bis(2-hydoxybenzylidene)acetone (HBB2), a dual activator of NRF2 and HSF1, protects against the development of UV irradiation-mediated cutaneous squamous cell carcinoma in mice. We further show that HBB2 is an inducer of autophagy. In cells, HBB2 increases the levels of the autophagy-cargo protein p62/sequestosome 1, and the lipidated form of microtubule-associated protein light chain 3 isoform B. Activation of autophagy by HBB2 is impaired in NRF2-deficient cells, which have reduced autophagic flux and low basal and induced levels of p62. Conversely, HSF1-deficient cells have increased autophagic flux under both basal as well as HBB2-induced conditions, accompanied by increased p62 levels. Our findings suggest that NRF2 and HSF1 have opposing roles during autophagy, and illustrate the existence of tight mechanistic links between the cellular stress responses.

KEAP1 inhibition is neuroprotective and suppresses the development of epilepsy

Hippocampal sclerosis is a common acquired disease that is a major cause of drug-resistant epilepsy. A mechanism that has been proposed to lead from brain insult to hippocampal sclerosis is the excessive generation of reactive oxygen species, and consequent mitochondrial failure. Here we use a novel strategy to increase endogenous antioxidant defences using RTA 408, which we show activates nuclear factor erythroid 2-related factor 2 (Nrf2, encoded by NFE2L2) through inhibition of kelch like ECH associated protein 1 (KEAP1) through its primary sensor C151. Activation of Nrf2 with RTA 408 inhibited reactive oxygen species production, mitochondrial depolarization and cell death in an in vitro model of seizure-like activity. RTA 408 given after status epilepticus in vivo increased ATP, prevented neuronal death, and dramatically reduced (by 94%) the frequency of late spontaneous seizures for at least 4 months following status epilepticus. Thus, acute KEAP1 inhibition following status epilepticus exerts a neuroprotective and disease-modifying effect, supporting the hypothesis that reactive oxygen species generation is a key event in the development of epilepsy.

Sulfhydryl-Reactive Phytochemicals as Dual Activators of Transcription Factors NRF2 and HSF1

Two central regulators, nuclear factor-erythroid 2 p45-related factor 2 (NRF2) and heat shock factor 1 (HSF1), control the KEAP1/NRF2/ARE pathway and the heat shock response, two essential cellular defense mechanisms. Both systems are highly inducible under conditions of stress. Many small molecules, including certain phytochemicals, such as isothiocyanates and phenylpropanoids, and/or their metabolites, have the capacity to induce the KEAP1/NRF2/ARE pathway. Recent results suggest that a common signal that is sensed through cysteine modification(s) within Kelch-like ECH-associated protein 1 (KEAP1) and HSF1, or possibly within a negative regulator of HSF1, is responsible for triggering both pathways. Celastrol, withaferin A, gedunin, curcumin, and sulforaphane are examples of structurally diverse phytochemicals with a common chemical signature: reactivity with sulfhydryl groups. This reactivity underlies their biological activities as multitarget agents for which protective effects have been documented in numerous animal models of human disease and which include induction of large networks of transcriptional programs regulated by transcription factors NRF2 and HSF1.

C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape

Numerous small molecules (termed inducers), many of which are electrophiles, upregulate cytoprotective responses and inhibit pro-inflammatory pathways by activating nuclear factor-erythroid 2 p45-related factor 2 (NRF2). Key to NRF2 activation is the ability to chemically modifying critical sensor cysteines in the main negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), of which C151, C273 and C288 are best characterized. This study aimed to establish the requirement for these cysteine sensor(s) for the biological activities of the most potent NRF2 activators known to date, the cyclic cyanoenones, some of which are in clinical trials. It was found that C151 in KEAP1 is the main cysteine sensor for this class of inducers, irrespective of molecular size or shape. Furthermore, in primary macrophage cells expressing C151S mutant KEAP1, at low concentrations, the tricyclic cyanoenone TBE-31 is inactive as an activator of NRF2 as well as an inhibitor of lipopolysaccharide-stimulated gene expression of the pro-inflammatory cytokines IL6 and IL1β. However, at high inducer concentrations, NRF2 activation proceeds in the absence of C151, albeit at a lower magnitude. Our findings highlight the intrinsic flexibility of KEAP1 and emphasize the critical importance of establishing the precise dose of NRF2 activators for maintaining on-target selectivity.