A mitochondrial iron-responsive pathway regulated by DELE1.

The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Cellular metabolic stress responses via organelles.

Metabolite fluctuations following nutrient metabolism or environmental stresses impact various intracellular signaling networks and stress responses to maintain cellular and organismal homeostasis. It has been shown that subcellular organelles, such as the endoplasmic reticulum, the Golgi apparatus, lysosomes and mitochondria serve as crucial hubs linking alterations in metabolite levels to cellular responses. This role is coordinated by molecular machineries that are associated with the lipid membranes of organelles, which sense the fluctuations in specific metabolites and activate the appropriate signaling and effector molecules. Moreover, recent studies have demonstrated that membraneless organelles, such as the nucleolus and stress granules, are involved in the metabolic stress response. Metabolite-induced post-translational modifications appear to play an important role in this process. Here, we review the molecular mechanisms of metabolite sensing and metabolite-mediated stress responses through membrane-bound and membraneless organelles in mammalian cells.

The Kelch repeat protein KLHDC10 regulates oxidative stress-induced ASK1 activation by suppressing PP5.

Reactive oxygen species (ROS)-induced activation of Apoptosis signal-regulating kinase 1 (ASK1) plays crucial roles in oxidative stress-mediated cell death through the activation of the JNK and p38 MAPK pathways. However, the regulatory mechanism of ASK1 in the oxidative stress response remains to be elucidated. Here, we identified the kelch repeat protein, Slim, as an activator of ASK1 through a Drosophila misexpression screen. We also performed a proteomics screen and revealed that Kelch domain containing 10 (KLHDC10), a mammalian ortholog of Slim, interacted with Protein phosphatase 5 (PP5), which has been shown to inactivate ASK1 in response to ROS. KLHDC10 bound to the phosphatase domain of PP5 and suppressed its phosphatase activity. Moreover, KLHDC10 was required for H(2)O(2)-induced sustained activation of ASK1 and cell death in Neuro2A cells. These findings suggest that Slim/KLHDC10 is an activator of ASK1, contributing to oxidative stress-induced cell death through the suppression of PP5.

Apoptosis Signal-regulating Kinase 1 (ASK1)-p38 Pathway-dependent Cytoplasmic Translocation of the Orphan Nuclear Receptor NR4A2 Is Required for Oxidative Stress-induced Necrosis.

p38 mitogen-activated protein kinases (MAPKs) play important roles in various cellular stress responses, including cell death, which is roughly categorized into apoptosis and necrosis. Although p38 signaling has been extensively studied, the molecular mechanisms of p38-mediated cell death are unclear. ASK1 is a stress-responsive MAP3K that acts as an upstream kinase of p38 and is activated by various stresses, such as oxidative stress. Here, we show that NR4A2, a member of the NR4A nuclear receptor family, acts as a necrosis promoter downstream of ASK1-p38 pathway during oxidative stress. Although NR4A2 is well known as a nucleus-localized transcription factor, we found that it is translocated into the cytosol after phosphorylation by p38. Because the phosphorylation site mutants of NR4A2 cannot rescue the cell death-promoting activity, ASK1-p38 pathway-dependent phosphorylation and subsequent cytoplasmic translocation of NR4A2 may be required for oxidative stress-induced cell death. In addition, NR4A2-mediated cell death does not depend on caspases and receptor-interacting protein 1 (RIP1)-RIP3 complex, suggesting that NR4A2 promotes an RIP kinase-independent necrotic type of cell death. Our findings may enable a more precise understanding of molecular mechanisms that regulate oxidative stress-induced and p38-mediated necrosis.

OMA1 clears traffic jam in TOM tunnel in mammals.

Using an engineered mitochondrial clogger, Krakowczyk et al. (https://doi.org/10.1083/jcb.202306051) identified the OMA1 protease as a critical component that eliminates import failure at the TOM translocase in mammalian cells, providing a novel quality control mechanism that is distinct from those described in yeast.

p38 MAPKs regulate the expression of genes in the dopamine synthesis pathway through phosphorylation of NR4A nuclear receptors.

In Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses.

Development and validation of the Japanese version of the Decisional Conflict Scale to investigate the value of pharmacists' information: a before and after study.

BACKGROUND: The information provided in patient-centered care and shared decision-making influences patients' concerns and adherence to treatment. In the decision-making process, patients experience decisional conflict. The Decisional Conflict Scale (DCS) is a 16-item, self-administered questionnaire consisting of 5 subscales developed to assess patients' decisional conflict. This study aimed to develop the Japanese version of the DCS and to clarify the influence of the information provided by pharmacists' on decisional conflict among patients with cancer. METHODS: We developed the Japanese version of the DCS by using the forward-backward translation method. One hundred patients who were recommended a new chemotherapy regimen were recruited. The psychometric properties of the Japanese DCS, including internal consistency, convergent validity, discriminant validity, and construct validity, were examined. We assessed the decisional conflict of patients before and after the pharmacists' provision of information. RESULTS: Ninety-four patients, predominately female, with an average age of 58.1 years were sampled. The scores on the 5 subscales of the DCS showed high internal consistency (Cronbach's alpha = 0.84-0.96). Multi-trait scaling analysis and cluster analysis showed strong validity. The mean total DCS score decreased significantly from 40.2 to 31.7 after patients received information from the pharmacists (p < 0.001, paired t-test). Scores on all 5 subscales, namely, uncertainty, informed, values clarity, support, and effective decision, also significantly improved (p < 0.001 for all categories, paired t-test). CONCLUSIONS: The psychometric properties of the Japanese version of the DCS are considered appropriate for it to be administered to patients with cancer. Pharmacists' provision of information was able to decrease decisional conflict among patients with cancer who were recommended a new chemotherapy regimen.

[Status of use and side effects of atovaquone for the treatment and prevention of pneumocystis pneumonia in HIV infected patients in Japan--from 1997 to 2012--The Clinical Study Group for AIDS Drugs, supported by Health and Labor Science Grants from the Ministry of Health, Labor and Welfare, Japan].

PURPOSE: Atovaquone is effective and well-tolerated for the treatment of mild or moderate Pneumocystis pneumonia (PCP) and the prevention of PCP. When atovaquone was not yet approved in Japan, it was supplied by the Clinical Study Group for AIDS Drugs, which has been supported by the Japan Health Science Foundation since 1997. We investigated the status of use and the reported side effects, since atovaquone has recently been approved and made available in Japan. METHOD: We retrospectively examined the application and adverse events associated with atovaquone use between January 1997 and March 2012. RESULTS: During this period, there were 721 new applications, increasing over time, with the highest rate of increase observed in recent years. Fifty-seven adverse events in 39 patients were reported. Drug eruption was the most common side effect (20 cases), followed by cytopenia (11 cases), fever (10 cases), and liver dysfunction (8 cases). Two deaths were reported (one with an unknown correlation, another with no comments provided). One case of liver dysfunction attributable to atovaquone was severe. In this case, the AST and ALT levels increased to 1,921 IU/L, and 1,062 IU/L, respectively on day 4 of atovaquone administration, but these levels improved after atovaquone discontinuation. No other severe side effects were reported. DISCUSSION: This study revealed that as in other countries, few side effects caused by atovaquone were reported in Japan. Moreover, there were no side effects unique to the Japanese population. However, caution is required when administering atovaquone, because a low incidence of severe atovaquone-induced liver dysfunction has been reported.

Adherence to anti-retroviral therapy, decisional conflicts, and health-related quality of life among treatment-naïve individuals living with HIV: a DEARS-J observational study.

BACKGROUND: Supporting people living with HIV using anti-retroviral therapy (ART) is important due to the requirement for strict medication adherence. To date, no data from longitudinal studies evaluating adherence by treatment-naïve people living with HIV are currently available. We investigated the adherence of treatment-naïve people living with HIV over time and examined the relationships among decisional conflicts, adherence, and health-related quality of life (HRQL). METHODS: The survey items included adherence (visual analogue scale [VAS]), decisional conflict (decisional conflict scale [DCS]), and HRQL (Medical Outcomes Study HIV Health Survey [MOS-HIV]). The DCS and MOS-HIV scores and the VAS and MOS scores were collected electronically at the ART initiation time point and at 4-, 24-, and 48-week post-treatment time points. RESULTS: A total of 215 participants were enrolled. The mean DCS score was 27.3 (SD, 0.9); 23.3% of participants were in the high-score and 36.7% in the low-score groups. The mean adherence rates at 4, 24, and 48 weeks were 99.2% (standard error [SE], 0.2), 98.4% (SE, 0.4), and 96.0% (SE, 1.2), respectively. The least-square means of the MOS-HIV for the DCS (high vs. low scores) were 64.4 vs. 69.2 for general health perceptions and 57.7 vs. 64.0 for HRQL, respectively. CONCLUSION: Adherence among treatment-naïve people living with HIV was maintained at a higher level, and HRQL tended to improve with ART. People with high levels of decisional conflict tended to have lower HRQL scores. Support for people living with HIV during ART initiation may be related to HRQL.

Mitochondrial phosphoglycerate mutase 5 uses alternate catalytic activity as a protein serine/threonine phosphatase to activate ASK1.

Phosphoglycerate mutase (PGAM) is an enzyme of intermediary metabolism that converts 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. Here, we discovered PGAM5 that is anchored in the mitochondrial membrane lacks PGAM activity and instead associates with the MAP kinase kinase kinase ASK1 and acts as a specific protein Ser/Thr phosphatase that activates ASK1 by dephosphorylation of inhibitory sites. Mutation of an active site His-105 in PGAM5 abolished phosphatase activity with ASK1 and phospho-Thr peptides as substrates. The Drosophila and Caenorhabditis elegans orthologs of PGAM5 also exhibit specific Ser/Thr phosphatase activity and activate the corresponding Drosophila and C. elegans ASK1 kinases. PGAM5 is unrelated to the other known Ser/Thr phosphatases of the PPP, MPP, and FCP families, and our results suggest that this member of the PGAM family has crossed over from small molecules to protein substrates and been adapted to serve as a specialized activator of ASK1.

Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay.

Guanine nucleotide-exchange factors (GEFs) activate the function of guanine nucleotide-binding proteins (G-proteins) by promoting the exchange of GDP for GTP on the latter. Here, we describe a protocol for in vitro measurements of the GEF activity of eukaryotic translation initiation factor 2B, eIF2B, toward its substrate eIF2. This protocol provides a relatively simple method for determining the eIF2B's GEF activity in crude cell extracts. The eIF2 heterotrimeric substrate, with phosphorylated or unphosphorylated eIF2α, is prepared by immunoprecipitation, following subsequent loading of a fluorescent BODIPY-FL dye-attached GDP. The exchange of the bound fluorescent GDP molecule for an unlabeled one on eIF2 promoted by eIF2B is monitored kinetically using a fluorescence microplate reader.

Discovery of bactericides as an acute mitochondrial membrane damage inducer.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Modulation of lysosomal function as a therapeutic approach for coronaviral infections.

The endo-lysosomal pathway plays an important role in pathogen clearance and both bacteria and viruses have evolved complex mechanisms to evade this host system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional regulator of lysosome biogenesis - TFEB - is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased siRNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. In particular, viral infection triggers marked PAK2 activation, which in turn, phosphorylates and primes TFEB for ubiquitin-mediated protein degradation. Deletion of either DCAF7 or PAK2 blocks viral-mediated TFEB degradation and protects against viral-induced cytopathic effects. We further derive a series of small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit viral-triggered TFEB degradation and demonstrate broad anti-viral activities including attenuating in vivo SARS-CoV-2 infection. Together, these results delineate a viral-triggered pathway that disables the endogenous cellular system that maintains lysosomal function and suggest that small molecule inhibitors of the E3 ubiquitin ligase DCAF7 represent a novel class of endo-lysosomal, host-directed, anti-viral therapies.

The coupling of translational control and stress responses.

The translation of messenger RNA (mRNA) into protein is a multistep process by which genetic information transcribed into an mRNA is decoded to produce a specific polypeptide chain of amino acids. Ribosomes play a central role in translation by coordinately working with various translation regulatory factors and aminoacyl-transfer RNAs. Various stresses attenuate the ribosomal synthesis in the nucleolus as well as the translation rate in the cytosol. To efficiently reallocate cellular energy and resources, mammalian cells are endowed with mechanisms that directly link the suppression of translation-related processes to the activation of stress adaptation programmes. This review focuses on the integrated stress response (ISR) and the nucleolar stress response (NSR) both of which are activated by various stressors and selectively upregulate stress-responsive transcription factors. Emerging findings have delineated the detailed molecular mechanisms of the ISR and NSR and expanded their physiological and pathological significances.

Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors.

Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

Hyperlipidaemia and IFNgamma/TNFalpha Synergism are associated with cholesterol crystal formation in Endothelial cells partly through modulation of Lysosomal pH and Cholesterol homeostasis.

BACKGROUND: Inflammation plays an important role in the development of cardiovascular disease (CVD). Patients with chronic inflammation diseases have high levels of inflammation and early fatal myocardial infarction due to early, unstable coronary plaques. Cholesterol crystals (CC) play a key role in atherogenesis. However, the underlying mechanisms of endothelial cell (EC)-derived CC formation are not well understood in chronic inflammation. METHODS: We utilized a combination of a mouse psoriasis model (K14-Rac1V12 mouse model) and human psoriasis patients to study the effect of inflammatory cytokines on CC formation in ECs. Lysosomal pH, alterations in lipid load and inflammatory proteins were evaluated as potential mechanisms linking inflammatory cytokines to CC formation. Coronary CT angiography was performed (n = 224) to characterize potential IFNγ and TNFα synergism on vascular diseases in vivo. FINDINGS: We detected CC presence in the aorta of K14-Rac1V12 mice on chow diet. IFNγ and TNFα were found to synergistically increase LDL-induced CC formation by almost 2-fold. There was an increase in lysosomal pH accompanied by a 28% loss in pH-dependent lysosomal signal and altered vATPaseV1E1 expression patterns. In parallel, we found that LDL+IFNγ/TNFα treatments increased free cholesterol content within EC and led to a decrease in SOAT-1 expression, an enzyme critically involved cholesterol homeostasis. Finally, the product of IFNγ and TNFα positively associated with early non-calcified coronary burden in patients with psoriasis (n = 224; ß = 0.28, p < 0.001). INTERPRETATION: Our results provide evidence that IFNγ and TNFα accelerate CC formation in endothelial cells in part by altering lysosomal pH and free cholesterol load. These changes promote early atherogenesis and contribute to understanding the burden of CVD in psoriasis. FUNDING: Funding was provided by the Intramural Research Program at NIH (NNM) and the National Psoriasis Foundation (NNM and YB).

Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B.

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the ß and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

The ASK1-MAP kinase signaling in ER stress and neurodegenerative diseases.

Accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) lumen induces ER stress. ER stress triggers the unfolded protein response (UPR), which includes the attenuation of general protein synthesis and the transcriptional activation of the genes encoding ER-resident chaperones and molecules involved in the ER-associated degradation (ERAD). The UPR coordinately reduces ER stress by restoration of the protein-folding capacity of the ER. However, severe and/or prolonged ER stress eventually leads cells to apoptosis. Several lines of evidence suggest that ER stress-induced apoptosis plays critical roles in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, polyglutamine (polyQ) diseases and amyotrophic lateral sclerosis (ALS). Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPKKK family that constitutes the JNK and p38 MAP kinase (MAPK) cascades, is activated by physiological and cytotoxic stresses and induces various stress responses including apoptosis. Recent studies have shown that the ASK1-MAPK cascades are involved in ER stress-induced apoptosis and in the neuronal cell death in some model systems of neurodegenerative diseases. This review highlights the current understanding of regulatory mechanisms of ASK1 with a special focus on the ER stress-dependent and -independent neuronal cell death in the context of neurodegenerative diseases.