Molecular Mechanism of Ethanol Fermentation Inhibition via Protein Tyrosine Nitration of Pyruvate Decarboxylase by Reactive Nitrogen Species in Yeast

Protein tyrosine nitration (PTN), in which tyrosine (Tyr) residues on proteins are converted into 3-nitrotyrosine (NT), is one of the post-translational modifications mediated by reactive nitrogen species (RNS). Many recent studies have reported that PTN contributed to signaling systems by altering the structures and/or functions of proteins. This study aimed to investigate connections between PTN and the inhibitory effect of nitrite-derived RNS on fermentation ability using the yeast Saccharomyces cerevisiae. The results indicated that RNS inhibited the ethanol production of yeast cells with increased intracellular pyruvate content. We also found that RNS decreased the activities of pyruvate decarboxylase (PDC) as a critical enzyme involved in ethanol production. Our proteomic analysis revealed that the main PDC isozyme Pdc1 underwent the PTN modification at Tyr38, Tyr157, and Tyr344. The biochemical analysis using the recombinant purified Pdc1 enzyme indicated that PTN at Tyr157 or Tyr344 significantly reduced the Pdc1 activity. Interestingly, the substitution of Tyr157 or Tyr344 to phenylalanine, which is no longer converted into NT, recovered the ethanol production under the RNS treatment conditions. These findings suggest that nitrite impairs the fermentation ability of yeast by inhibiting the Pdc1 activity via its PTN modification at Tyr157 and Tyr344 of Pdc1.

A Ratiometric Theranostic System for Visualization of ONOO– Species and Reduction of Drug-Induced Hepatotoxicity

Peroxynitrite (ONOO–) is a potent reactive nitrogen species that plays a critical mediator in liver injury elicited by drugs such as acetaminophen (APAP). At a therapeutic dosage, most APAP is...

Clotting Dysfunction in Sepsis: A Role for ROS and Potential for Therapeutic Intervention

Sepsis is regarded as one of the main causes of death among the critically ill. Pathogen infection results in a host-mediated pro-inflammatory response to fight infection; as part of this response, significant endogenous reactive oxygen (ROS) and nitrogen species (RNS) production occurs, instigated by a variety of sources, including activated inflammatory cells, such as neutrophils, platelets, and cells from the vascular endothelium. Inflammation can become an inappropriate self-sustaining and expansive process, resulting in sepsis. Patients with sepsis often exhibit loss of aspects of normal vascular homeostatic control, resulting in abnormal coagulation events and the development of disseminated intravascular coagulation. Diagnosis and treatment of sepsis remain a significant challenge for healthcare providers globally. Targeting the drivers of excessive oxidative/nitrosative stress using antioxidant treatments might be a therapeutic option. This review focuses on the association between excessive oxidative/nitrosative stress, a common feature in sepsis, and loss of homeostatic control at the level of the vasculature. The literature relating to potential antioxidants is also described.

ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.