Redox-Regulation of α-Globin in Vascular Physiology

Interest in the structure, function, and evolutionary relations of circulating and intracellular globins dates back more than 60 years to the first determination of the three-dimensional structure of these proteins. Non-erythrocytic globins have been implicated in circulatory control through reactions that couple nitric oxide (NO) signaling with cellular oxygen availability and redox status. Small artery endothelial cells (ECs) express free α-globin, which causes vasoconstriction by degrading NO. This reaction converts reduced (Fe2+) α-globin to the oxidized (Fe3+) form, which is unstable, cytotoxic, and unable to degrade NO. Therefore, (Fe3+) α-globin must be stabilized and recycled to (Fe2+) α-globin to reinitiate the catalytic cycle. The molecular chaperone α-hemoglobin-stabilizing protein (AHSP) binds (Fe3+) α-globin to inhibit its degradation and facilitate its reduction. The mechanisms that reduce (Fe3+) α-globin in ECs are unknown, although endothelial nitric oxide synthase (eNOS) and cytochrome b5 reductase (CyB5R3) with cytochrome b5 type A (CyB5a) can reduce (Fe3+) α-globin in solution. Here, we examine the expression and cellular localization of eNOS, CyB5a, and CyB5R3 in mouse arterial ECs and show that α-globin can be reduced by either of two independent redox systems, CyB5R3/CyB5a and eNOS. Together, our findings provide new insights into the regulation of blood vessel contractility.

Extracellular Calcium Receptor as a Target for Glutathione and Its Derivatives

Extracellular glutathione (GSH) and oxidized glutathione (GSSG) can modulate the function of the extracellular calcium sensing receptor (CaSR). The CaSR has a binding pocket in the extracellular domain of CaSR large enough to bind either GSH or GSSG, as well as the naturally occurring oxidized derivative L-cysteine glutathione disulfide (CySSG) and the compound cysteinyl glutathione (CysGSH). Modeling the binding energies (ΔG) of CySSG and CysGSH to CaSR reveals that both cysteine derivatives may have greater affinities for CaSR than either GSH or GSSG. GSH, CySSG, and GSSG are found in circulation in mammals and, among the three, CySSG is more affected by HIV/AIDs and aging than either GSH or GSSG. The beta-carbon linkage of cysteine in CysGSH may model a new class of calcimimetics, exemplified by etelcalcetide. Circulating glutathionergic compounds, particularly CySSG, may mediate calcium-regulatory responses via receptor-binding to CaSR in a variety of organs, including parathyroids, kidneys, and bones. Receptor-mediated actions of glutathionergics may thus complement their roles in redox regulation and detoxification. The glutathionergic binding site(s) on CaSR are suggested to be a target for development of drugs that can be used in treating kidney and other diseases whose mechanisms involve CaSR dysregulation.

Environmental Tuning of Homologs of the Orange Carotenoid Protein-Encoding Gene in the Cyanobacterium Fremyella diplosiphon

The orange carotenoid protein (OCP) family of proteins are light-activated proteins that function in dissipating excess energy absorbed by accessory light-harvesting complexes, i.e., phycobilisomes (PBSs), in cyanobacteria. Some cyanobacteria contain multiple homologs of the OCP-encoding gene (ocp). Fremyella diplosiphon, a cyanobacterium studied for light-dependent regulation of PBSs during complementary chromatic acclimation (CCA), contains several OCP homologs – two full-length OCPs, three Helical Carotenoid Proteins (HCPs) with homology to the N-terminus of OCP, and one C-terminal domain-like carotenoid protein (CCP) with homology to the C-terminus of OCP. We examined whether these homologs are distinctly regulated in response to different environmental factors, which could indicate distinct functions. We observed distinct patterns of expression for some OCP, HCP, and CCP encoding genes, and have evidence that light-dependent aspects of ocp homolog expression are regulated by photoreceptor RcaE which controls CCA. RcaE-dependent transcriptional regulator RcaC is also involved in the photoregulation of some hcp genes. Apart from light, additional environmental factors associated with cellular redox regulation impact the mRNA levels of ocp homologs, including salt, cold, and disruption of electron transport. Analyses of conserved sequences in the promoters of ocp homologs were conducted to gain additional insight into regulation of these genes. Several conserved regulatory elements were found across multiple ocp homolog promoters that potentially control differential transcriptional regulation in response to a range of environmental cues. The impact of distinct environmental cues on differential accumulation of ocp homolog transcripts indicates potential functional diversification of this gene family in cyanobacteria. These genes likely enable dynamic cellular protection in response to diverse environmental stress conditions in F. diplosiphon.

Free radicals and oxidative stress: signaling mechanisms, redox basis for human diseases, and cell cycle regulation

: Free radical contained one or more unpaired electrons in its valence shell, thus making it unstable, short-lived and highly reactive specie. Excessive generation of these free radicals ultimately leads to oxidative stress causing oxidation and damage to significant macromolecules in the living system and essentially disrupting signal transduction pathways and antioxidants equilibrium. At lower concentrations, ROS serves as “second messengers” influencing many physiological processes in the cell. However, at higher concentrations beyond cell capacity causes oxidative stress, which contributes to much human pathology such as diabetes, cancer, Parkinson’s disease, cardiovascular diseases, cataract, asthma, hypertension, atherosclerosis, arthritis and Alzheimer’s disease. Signaling pathways such as NF-κB, MAPKs, PI3K/Akt/ mTOR and Keap1-Nrf2-ARE modulates the detrimental effects of oxidative stress by increasing the expression of cellular antioxidant defenses, phase II detoxification enzymes and decreased production of ROS. Free radicals such as H2O2 are indeed needed for the advancement of cell cycle as these molecules influences DNA, proteins and enzymes in the cell cycle pathway. In the course of cell cycle progression, the cellular redox environment becomes more oxidized moving from G1 phase, becomes higher in G2/M and moderate in S phase. Signals in the form of an increase in cellular pro-oxidant levels are required and these signals are often terminated by a rise in the amount of antioxidants and MnSOD with a decrease in the level of cyclin D1 proteins. Therefore, understanding the mechanism of cell cycle redox regulation will help in therapy of many diseases.

The Impact of the hAPP695SW Transgene and Associated Amyloid-β Accumulation on Murine Hippocampal Biochemical Pathways

Background: Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) peptide in the brain. Objective: Gain a better insight into alterations in major biochemical pathways underlying AD. Methods: We compared metabolomic profiles of hippocampal tissue of 20-month-old female Tg2576 mice expressing the familial AD-associated hAPP695SW transgene with their 20-month-old wild type female littermates. Results: The hAPP695SW transgene causes overproduction and accumulation of Aβ in the brain. Out of 180 annotated metabolites, 54 metabolites differed (30 higher and 24 lower in Tg2576 versus wild-type hippocampal tissue) and were linked to the amino acid, nucleic acid, glycerophospholipid, ceramide, and fatty acid metabolism. Our results point to 1) heightened metabolic activity as indicated by higher levels of urea, enhanced fatty acid β-oxidation, and lower fatty acid levels; 2) enhanced redox regulation; and 3) an imbalance of neuro-excitatory and neuro-inhibitory metabolites in hippocampal tissue of aged hAPP695SW transgenic mice. Conclusion: Taken together, our results suggest that dysregulation of multiple metabolic pathways associated with a concomitant shift to an excitatory-inhibitory imbalance are contributing mechanisms of AD-related pathology in the Tg2576 mouse.

Proteomic Analysis of Subchronic Furan Exposure in the Liver of Male Fischer F344 Rats

Furan is a volatile compound formed during the thermal processing of foods. Chronic exposure has been shown to cause cholangiocarcinoma and hepatocellular tumors in rodent models. We conducted a 90 day subchronic study in Fisher 344 rats exposed to various doses by gavage to determine the NOAEL. Previous reports have outlined changes in the liver using gross necropsy examination, histopathology, clinical biochemistry, hematology, immunohistochemistry, and toxicogenomics. The data revealed that males were more sensitive than females. The focus of this study was to evaluate the toxicoproteomic changes by 2-dimensional differential in gel electrophoresis followed by mass spectrometry analysis. To compliment previous studies, protein expression changes were evaluated of male animals after 90 days of exposure to doses of 0, 0.03, 0.5, and 8.0 mg/kg bw/d. Significant statistical treatment-related changes compared to the controls identified 45 protein spots containing 38 unique proteins. Proteins identified are implicated in metabolism, redox regulation, protein folding/proteolysis as well as structural and transport proteins. At lower doses, multiple cytoprotective pathways are activated to maintain a homeostasis but ultimately the loss of protein function and impairment of several pathways could lead to adverse health effects at higher doses of furan administration.

Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.

Quantitative Redox Proteomics Revealed Molecular Mechanisms of Salt Tolerance in the Roots of Sugar Beet Monomeric Addition line M14

Background: Salt stress is often associated with excessive production of reactive oxygen species (ROS). Oxidative stress caused by the accumulation of ROS is a major factor that negatively affects crop growth and yield. Root is the primary organ that senses and transmits the salt stress signal to the whole plant. How oxidative stress affect redox sensitive proteins in the roots is not known.Results: In this study, the redox proteome of sugar beet M14 roots under salt stress was investigated. Using iTRAQ reporters, we determined that salt stress caused significant changes in the abundance of many proteins (2305 at 20 min salt stress and 2663 at 10 min salt stress). Using iodoTMT reporters, a total of 95 redox proteins were determined to be responsive to salt stress after normalizing again total protein level changes. Notably, most of the differential redox proteins were involved in metabolism, ROS homeostasis, and stress and defense, while a small number play a role in transport, biosynthesis, signal transduction, transcription and photosynthesis. Transcription levels of 14 genes encoding the identified redox proteins were analyzed using qRT-PCR. All the genes were induced by salt stress at the transcriptional level. Conclusions: Based on the redox proteomics results, we construct a map of the regulatory network of M14 root redox proteins in response to salt stress. This study further refines the molecular mechanism of salt resistance at the level of protein redox regulation.

Role of Mitochondria in Physiology of Chondrocytes and Diseases of Osteoarthritis and Rheumatoid Arthritis

Purpose of Review Mitochondria are recognized to be one of the most important organelles in chondrocytes for their role in triphosphate (ATP) generation through aerobic phosphorylation. Mitochondria also participate in many intracellular processes involving modulating reactive oxygen species (ROS), responding to instantaneous hypoxia stress, regulating cytoplasmic transport of calcium ion, and directing mitophagy to maintain the homeostasis of individual chondrocytes. Designs To summarize the specific role of mitochondria in chondrocytes, we screened related papers in PubMed database and the search strategy is ((mitochondria) AND (chondrocyte)) AND (English [Language]). The articles published in the past 5 years were included and 130 papers were studied. Results In recent years, the integrity of mitochondrial structure has been regarded as a prerequisite for normal chondrocyte survival and defect in mitochondrial function has been found in cartilage-related diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA). However, the understanding of mitochondria in cartilage is still largely limited. The mechanism on how the changes in mitochondrial structure and function directly lead to the occurrence and development of cartilage-related diseases remains to be elusive. Conclusion This review aims to summarize the role of mitochondria in chondrocytes under the physiological and pathological changes from ATP generation, calcium homeostasis, redox regulation, mitophagy modulation, mitochondria biogenesis to immune response activation. The enhanced understanding of molecular mechanisms in mitochondria might offer some new cues for cartilage remodeling and pathological intervention.