N-Terminally arginylated ubiquitin is attached to histone H2A by RING1B E3 ligase in human cells.

Ubiquitin (Ub) is highly conserved in all eukaryotic organisms and begins at the N-terminus with Met and Gln. Our recent research demonstrates that N-terminally (Nt-) arginylated Ub can be produced in the yeast Saccharomyces cerevisiae. However, the existence of Nt-arginylated Ub in multicellular organisms remains unknown. Here we explore the mechanism for creating Nt-arginylated Ub using human embryonic kidney HEK293 cells that express various Nt-modified Ubs. We found that Gln-starting Q-Ub was converted into Glu-starting E-Ub by NTAQ1 Nt-deamidase and subsequently Nt-arginylated by ATE1 arginyltransferase in HEK293 cells. We also found that the resulting Arg-Glu-starting RE-Ub was mainly deposited on the Lys119 residue of histone H2A. Furthermore, RING1B E3 Ub ligase mediated the attachment of RE-Ub to H2A. These findings reveal a previously unknown type of histone ubiquitylation which greatly increases the combinatorial complexity of histone and ubiquitin codes.

The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis.

Ferroptosis is a unique form of cell death caused by excessive iron-dependent lipid peroxidation. The level of the anabolic reductant NADPH is a biomarker of ferroptosis sensitivity. However, specific regulators that detect cellular NADPH levels, thereby modulating downstream ferroptosis cascades, are largely unknown. We show here that the transmembrane endoplasmic reticulum MARCHF6 E3 ubiquitin ligase recognizes NADPH through its C-terminal regulatory region. This interaction upregulates the E3 ligase activity of MARCHF6, thus downregulating ferroptosis. We also found that MARCHF6 mediates the degradation of the key ferroptosis effectors ACSL4 and p53. Furthermore, inhibiting ferroptosis rescued the growth of MARCHF6-deficient tumours and peri-natal lethality of Marchf6-/- mice. Together, these findings identify MARCHF6 as a previously unknown NADPH sensor in the ubiquitin system and a crucial regulator of ferroptosis.

Quercetin and chloroquine synergistically kill glioma cells by inducing organelle stress and disrupting Ca2+ homeostasis.

Glioblastoma (GBM) remains one of the most uncompromising cancers, with a median survival of 15 months among those receiving maximal therapy. Therefore, new effective approaches are urgently required for the treatment of GBM. In this study, we show that combined treatments with the flavonoid quercetin and chloroquine (CQ), which is a lysosomotropic agent with antimalarial activity, synergistically induce caspase-independent cell death in malignant glioma cells. The combination of quercetin and CQ triggered excessive expansion of autolysosomes and lysosomes due to overloading with undigested cellular components and protein aggregates, leading to cell death, whereas quercetin alone increased autophagic flux. These results suggest that CQ-mediated lysosomal inhibition prolongs quercetin-mediated autophagic flux, resulting in autophagic catastrophe and severe endoplasmic reticulum (ER) stress. Additionally, we found that 1,4,5-triphosphate receptor (IP3R)-mediated Ca2+ release from the ER and the following mitochondrial uniporter (MCU)-mediated Ca2+ influx into mitochondria as well as ROS generation are critically involved in the cytotoxicity by this combination. Collectively, the lysosomal defects induced by quercetin plus CQ may trigger the stress to both the ER and mitochondria and consequently their functional defects, contributing to glioma cell death. The combination of quercetin and CQ may be an effective therapeutic option for GBM.

Diagnostic performance of initial serum lactate for predicting bacteremia in female patients with acute pyelonephritis.

OBJECTIVES: The purpose of the present study was to investigate the diagnostic value of lactate for predicting bacteremia in female patients with acute pyelonephritis (APN). METHODS: We conducted a retrospective study of female patients with APN who visited the study hospital emergency department. The demographics, comorbidities, physiologies, and laboratory variables including white blood cell count and segmented neutrophil count, C-reactive protein, and initial serum lactate levels were collected and analyzed to identify associations with the presence of bacteremia. RESULTS: During the study period, a total of 314 patients were enrolled. One hundred twenty-three patients (39.2%) had bacteremia. Escherichia coli was the most frequent pathogen. Logistic regression analysis demonstrated that the lactate level was independently associated with the presence of bacteremia (odds ratio, 1.39 [95% confidence interval, 1.08-1.78]). The C-statistic of the lactate level was 0.67 (95% CI, 0.60-0.73). At a cutoff value of 1.4mmol/L, the lactate level predicted bacteremia with a sensitivity (53.7%), specificity (72.3%), positive predictive value (55.5%), negative predictive value (70.8%), positive likelihood ratio (1.93), and negative likelihood ratio (0.64). CONCLUSION: The initial serum lactate level showed poor discriminative performance for predicting bacteremia in female patients with APN.