Alpha-Ketoglutarate and 5-HMF: A Potential Anti-Tumoral Combination against Leukemia Cells

We have recently shown that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) might have anti-tumoral potential due to its antioxidative activities. The question arises if these substances have caspase-3- and apoptosis-activating effects on the cell proliferation in Jurkat and HF-SAR cells. Antioxidative capacity of several combined aKG + 5-HMF solution was estimated by cigarette smoke radical oxidized proteins of fetal calf serum (FCS) using the estimation of carbonylated proteins. The usage of 500 µg/mL aKG + 166.7 µg/mL 5-HMF showed the best antioxidative capacity to inhibit protein modification of more than 50% compared to control measurement. A Jurkat cell line and human fibroblasts (HF-SAR) were cultivated in the absence or presence of combined AKG + 5-HMF solutions between 0 µg/mL aKG + 0 µg/mL 5-HMF and different concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Aliquots of Jurkat cells were tested for cell proliferation, mitochondrial activity, caspase activity, apoptotic cells and of the carbonylated protein content as marker of oxidized proteins in cell lysates after 24, 48, and 72 h of incubation. The combined solutions of aKG + 5-HMF were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time, with the greatest reductions using 500 µg/mL aKG + 166.7 µg/mL 5-HMF after 24 h of incubation compared to 24 h with the control (22,832 cells vs. 32,537 cells), as well as after 48 h (21,243 vs. 52,123 cells) and after 72 h (23,224 cells). Cell growth was totally inhibited by the 500 µg/mL AKG + 166.7 µg/mL solution between 0 and 72 h of incubation compared to 0 h of incubation for the control. The mitochondrial activity measurements supported the data on cell growth in Jurkat cells: The highest concentration of 500 µg/mL aKG + 166.7 µg/mL 5-HMF was able to reduce the mitochondrial activity over 24 h (58.9%), 48 h (28.7%), and 72 h (9.9%) of incubation with Jurkat cells compared not only to the control incubation, but also to the concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 375 µg/mL aKG 125 µg/mL 5-HMF, which were able to significantly reduce the mitochondrial activity after 48 h (28.7% or 35.1%) and 72 h (9.9% or 18.2%) compared to 24 h with the control (100%). A slight increase in cell proliferation was found in HF-SAR using the highest concentration (500 µg/mL aKG + 166.7 µg/mL 5-HMF) between 0 h and 72 h incubation of 140%, while no significant differences were found in the mitochondrial activity of HF-SAR in the absence or presence of several combined aKG + 5-HMF solutions. The solutions with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF showed a significantly higher caspase activity (51.6% or 13.5%) compared to the control (2.9%) in addition to a higher apoptosis rate (63.2% or 31.4% vs. control: 14.9%). Cell lysate carbonylated proteins were significantly higher in Jurkat cells compared to HF-SAR cells (11.10 vs. 2.2 nmol/mg). About 72 h incubation of Jurkat cells with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF reduced significantly the carbonylated protein content down to 5.55 or 7.44 nmol/mg whereas only the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution showed a significant reduction of carbonylated proteins of HF-SAR (1.73 nmol/mg).

Immunoproteasome Function in Normal and Malignant Hematopoiesis

The ubiquitin–proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers.

Feeding oxidized chicken byproduct meal impacts digestibility more than performance and oxidative status in nursery pigs

Rendered products from the meat industry provide quality proteins in diets for companion animals. These proteins are exposed to extreme temperatures during processing leading to the potential for decreased diet digestibility and subsequent growth performance. While this would impact production efficiency in livestock species, oxidized ingredients in companion animal diets may impact health and longevity. The objective of this study was to determine the extent to which a feedstuff containing oxidized protein and lipid affect diet digestibility, growth performance, and oxidative stress in nursery pigs. A total of 56 male pigs (21 d of age, initial body weight 5.51 ± 0.65 kg) were randomly assigned to one of the four dietary treatments in a 2 × 2 factorial arrangement with two levels of heat and two levels of antioxidant (AOX). Diets were fed for 35 d and growth performance was measured, while total tract digestibility and nitrogen (N) balance was determined during the trial on day 18–20. Blood plasma was collected on day 34 and jejunum, colon, and liver tissues were collected on day 35 to analyze for markers of oxidative stress. Average daily feed intake (ADFI) was reduced in pigs fed diets without AOXs (P = 0.02). Additionally, pigs consuming diets containing heated chicken byproduct (CBP) meal had decreased gain:feed (GF; P = 0.02). There was an interaction between heat and AOX (P = 0.02) where heating CBP reduced N digestibility in the presence of an AOX but did not have an impact when AOX was not present. The removal of AOX resulted in reduced GE digestibility (P < 0.01). Dry matter (P < 0.01), ash (P < 0.01), and protein (P < 0.01) digestibility were reduced (P < 0.01) as a result of heating. Furthermore, heating (P =0.01) as well as absence of AOX (P =0.01) resulted in reduced digestible energy. No difference was detected in N retention suggesting that oxidation reduces digestibility but has no impact on N utilization. This is supported by the fact that systemic oxidative stress was not consistently affected by heating or AOX inclusion. These results suggest that feeding pigs CBP containing oxidized proteins and lipids did not induce oxidative stress. However, feeding young pigs CBP containing oxidized proteins and lipids did result in reduced energy and nutrient digestibility as well as negatively affected feed efficiency. Because CBP is commonly used in companion animal diets, it is reasonable to revisit their impacts on those species.

161 Dietary oxidized protein result in growth and histology differences in pigs

Rendered by-products are common feedstuffs in diets. During processing, these sources have potential to become oxidized. While most research on oxidative stress has focused on dietary oxidized lipids, little research has been done in dietary oxidized proteins and the potential to induce oxidative stress. Recent studies have shown consumption of dietary oxidized proteins resulted in shifts in oxidative status. The objective of this study was to determine the effects of dietary oxidized protein on oxidative stress in pigs as a model for canines. Fifty-six, 3 weeks old pigs (5.55 ± 0.71 Kg) were fed 4 dietary treatments each varying in dietary oxidized protein. Each treatment was fed the same diet, with the exception of the degree of oxidation in chicken by-product meal which was included in the diet at 23 percent to represent typical inclusion levels of a companion animal diet. Oxidation was induced by 2 levels of heating and 2 levels of antioxidants (mixed tocopherol), in a 2 x 2 factorial design. A significant antioxidant effect was observed in villi height (P-value < 0.05) where pigs fed diets without antioxidant had increased villi height (674 μm) compared to those fed diets with antioxidants (576 μm). Additionally, pigs fed diets without antioxidant had significantly greater villi:crypt (3.22) compared to those fed diets with antioxidants (2.84 ; P-value < 0.05). Additionally, feed efficiency measured by gain:feed, was significantly decreased in pigs fed heated protein (0.65) compared to not heated (0.69; P-value < 0.05). Results from this study are consistent with previous studies (Dibner, et al., 1996). Future studies should examine the effects of feeding oxidized proteins in regard to oxidative status, such as markers of protein, lipid, DNA, and antioxidative enzyme damage. In conclusion, dietary oxidized protein can reduce feed efficiency in pigs.

Susceptibility of Protein Methionine Oxidation in Response to Hydrogen Peroxide Treatment–Ex Vivo Versus In Vitro: A Computational Insight

Methionine oxidation plays a relevant role in cell signaling. Recently, we built a database containing thousands of proteins identified as sulfoxidation targets. Using this resource, we have now developed a computational approach aimed at characterizing the oxidation of human methionyl residues. We found that proteins oxidized in both cell-free preparations (in vitro) and inside living cells (ex vivo) were enriched in methionines and intrinsically disordered regions. However, proteins oxidized ex vivo tended to be larger and less abundant than those oxidized in vitro. Another distinctive feature was their subcellular localizations. Thus, nuclear and mitochondrial proteins were preferentially oxidized ex vivo but not in vitro. The nodes corresponding with ex vivo and in vitro oxidized proteins in a network based on gene ontology terms showed an assortative mixing suggesting that ex vivo oxidized proteins shared among them molecular functions and biological processes. This was further supported by the observation that proteins from the ex vivo set were co-regulated more often than expected by chance. We also investigated the sequence environment of oxidation sites. Glutamate and aspartate were overrepresented in these environments regardless the group. In contrast, tyrosine, tryptophan and histidine were clearly avoided but only in the environments of the ex vivo sites. A hypothetical mechanism of methionine oxidation accounts for these observations presented.

Efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins

The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (β1–β2–β5), the immunoproteasome (β1i–β2i–β5i) and the two intermediate proteasomes (β1–β2–β5i and β1i–β2–β5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c-­myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three β5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, β5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three β5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.

Role of the anionic component in manifestation of effects of organic lithium salts on oxidative modification of blood proteins and lipids in patients with depressive disorders

Введение. Литиевые соли широко используются в психиатрии. Изучение новых свойств солей лития с различными анионными компонентами является важной задачей. Цель исследования - изучение in vitro эффектов солей лития, содержащих разные органические анионные компоненты, на модификацию белков и липидов плазмы крови пациентов с депрессивными расстройствами (ДР). Методика. Инкубацию крови пациентов ДР с солями лития (аскорбат лития, сукцинат лития и пируват лития) проводили в течение 1 ч при 37 °С. В качестве окислительного агента использовали 0,5% этанол. Конечная концентрация в пробах ионов лития составляла 1,2 мМ. В плазме крови определяли количество окисленных белков по уровню карбонилированных белков (КБ) с использованием 2,4-динитрофенилгидразина, продукты перекисного окисления липидов - по содержанию продуктов, взаимодействующих с тиобарбитуровой кислотой (ТБК-реактивных продуктов, ТБК-РП). Результаты. У пациентов с ДР уровень КБ повышался по сравнению с контролем (здоровые лица), а концентрация ТБК-РП не отличалась от нормы. Инкубация крови здоровых лиц с 0,5%-ным этанолом приводила к увеличению содержания КБ и ТБК-РП плазмы, на уровень окисленных белков и липидов плазмы крови пациентов ДР в тех же условиях этанол влияния не оказывал. Аскорбат лития и пируват лития в присутствии этанола увеличивал уровень КБ в плазме крови пациентов ДР, сукцинат лития в этих условиях на КБ значимого эффекта не оказывал. На уровень ТБК-РП плазмы крови пациентов ДР все исследуемые соединения влияния не оказывали. В крови здоровых доноров соли лития проявляли антиоксидантный эффект, повышая устойчивость макромолекул плазмы к токсическому (окислительному) действию этанола. Заключение. Исследуемые соли лития в условиях эксперимента с применением в качестве окислительного агента этанола оказывали антиоксидантное действие на белки и липиды плазмы крови здоровых лиц. Соли лития, имеющие в своем составе анионы аскорбата или пирувата, проявляли прооксидантный эффект в образцах крови пациентов с депрессивными расстройствами, усиливая окислительное действие этанола на белки, но не на липиды плазмы. Таким образом, соли лития оказывают различное действие на окислительную модификацию белков и липидов крови здоровых лиц и пациентов депрессивными расстройствами in vitro, при этом анионный компонент соли играет важную роль в проявлении эффектов соединений. Introduction. Lithium salts are widely used in psychiatry. Studying new properties of lithium salts with various anionic components is important. The aim of this study was to compare in vitro effects of lithium salts containing different organic anionic components on oxidative modification of plasma proteins and lipids in patients with depressive disorders (DD). Methods. Blood of DD patients was incubated with lithium salts (lithium ascorbate, lithium succinate, and lithium pyruvate) at 37 °C for 1 hour. Ethanol was used as an oxidizing agent. Final concentrations of lithium ions and ethanol in a sample were 1.2 mM and 0.5%, respectively. Plasma content of oxidized proteins was determined by the content of carbonylated proteins (CP) using 2,4-dinitrophenylhydrazine. Lipid peroxidation products were determined by the content of thiobarbituric acid-reactive products (TBA-RP). Results. In DD patients, concentration of CP was increased compared to the control (healthy subjects) whereas concentration of TBA-RP did not differ from the control. Incubation of blood from healthy individuals with 0.5% ethanol resulted in increases in both the CP and TBA-RP whereas ethanol did not exert any effect on concentrations of oxidized proteins and TBA-RP in DD patients in the same conditions. Lithium ascorbate and lithium pyruvate in the presence of ethanol increased the CP concentration in blood from DD patients; lithium succinate left unchanged the CP concentration in these conditions. None of the studied compounds had any effect on TBA-RP in plasma of DD patients. In blood of healthy donors, lithium salts exerted an antioxidant effect increasing the resistance of plasma macromolecules to the toxic (oxidative) action of ethanol. Conclusion. The studied lithium salts produced an antioxidant effect in vitro on ethanol-induced oxidative modification of proteins and lipids in blood from healthy individuals. In blood samples from DD patients, lithium salts containing ascorbate or pyruvate anions showed pro-oxidant properties by potentiating the oxidative effect of ethanol on plasma proteins but not lipids. Therefore, lithium salts differently influence in vitro oxidative modification of proteins and lipids in blood of healthy people and DD patients with the anionic component of lithium salt playing an important role in manifestation of effects of these compounds.

P0029GLUCOSYLCERAMIDE SYNTHASE INHIBITION PRESERVES MITOCHONDRIAL FUNCTION AND REDUCES REACTIVE OXYGEN DAMAGE IN THE JCK MOUSE MODEL OF POLYCYSTIC KIDNEY DISEASE

Background and Aims Polycystic kidney disease (PKD) is characterized by the formation and progressive growth of renal cysts ultimately leading to end-stage renal disease (ESRD). Previous work has shown that treatment of PKD mouse models with a glucosylceramide synthase inhibitor (GCSi) inhibits glycosphingolipid metabolism and blocks disease progression. Clinical trials to determine the efficacy of GCSi treatment for ADPKD are currently ongoing. ADPKD patient kidneys, as well as kidneys from in vivo mouse models of PKD, display defects in mitochondrial morphology and function, leading to reactive oxygen species (ROS) generation. Functional defects include decreased fatty acid oxidation and increased glycolysis that can promote cellular proliferation. To determine if glucosylceramide synthase inhibition ameliorates mitochondrial dysfunction, we assessed the impact of GCSi treatment on mitochondrial formation and function in the jck mouse model of PKD. Method Twenty-six-day old WT or jck mice were treated with vehicle or 60 mg/kg GCSi (Genz667161) in feed for 38 days prior to tissue harvest. Kidney mRNA expression was measured using RT-PCR. Kidney protein levels were measured by western blot. Mitochondrial DNA content was measured using real-time PCR. Oxidized DNA was detected by immunohistochemistry using anti-8OHdG antibodies. Oxidized proteins were measured using the Oxyblot system (manufacturer). Results Reduced cystogenesis following GCSi treatment was associated with preserved mitochondrial number in jck mice as evidenced by increased levels of mitochondrial DNA, increased mitochondrial proteins (Tom20, VDAC, SirT3, and MT-Co1), and elevated mRNA expression (Nd3, Cox3, and Atp8). Stabilization of mitochondrial number was accompanied by activation of pathways that promote mitochondrial biogenesis, including increased PGC1α protein and Ppargc1a and Tfam1. Downregulation of several antioxidant genes (Gpx6, Gstk1, Prdx3, and Sod2) were observed in untreated jck samples. Consistent with this, levels of oxidized DNA and oxidized proteins were increased in untreated jck samples. Treatment with GCSi partially reversed the downregulation of antioxidant genes and decreased the levels of oxidized DNA and proteins to near WT levels in jck tissues. We then compared the jck model to tissues derived from ADPKD patients. Western blots demonstrate a reduction in electron transport chain (CxI-CxV) and mitochondrial outer-membrane markers in both untreated jck and ADPKD samples compared to controls. Similarly, the mitochondrial genes Nd1, Nd2, Cox2, and Atp6 were reduced in untreated jck kidneys and ADPKD samples relative to controls. As previously mentioned, we observed a generalized loss of antioxidant gene expression in untreated jck kidneys. Consistent with this, there were increased levels of both oxidized DNA and oxidized protein in untreated jck samples; this increase in DNA and protein oxidation was mirrored in ADPKD patient samples. Conclusion Mitochondrial number and function were reduced in both untreated jck mouse kidneys and human ADPKD samples. Increased oxidative stress, as evidenced by increased levels of oxidized DNA and proteins, is evident in both jck and ADPKD tissues. In vivo treatment with a GCSi effectively reversed the observed increase in oxidative stress and inhibited disease progression in the jck model of ADPKD. Our data demonstrate that reduced kidney cyst growth following GCSi treatment is associated with preserved mitochondrial formation and function.

Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

ABSTRACT Preexposure to a low concentration of H2O2 significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H2O2. However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 μM H2O2 and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn2+-coordinated Cys139 and Cys142 mutations eliminated the H2O2 oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn2+ in H2O2-treated PerR, demonstrating that cysteine oxidation results in Zn2+ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn2+-bound PerR protein (PerR:Zn,Mn) was abolished by H2O2 treatment but was restored by dithiothreitol reduction, verifying that H2O2 inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H2O2-concentration-induced adaptation of S. oligofermentans to a higher H2O2 concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H2O2 concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H2O2 defense strategy employed by catalase-negative streptococci in Mn2+-rich cellular environments. IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.