The Role of Stress-Induced O-GlcNAc Protein Modification in the Regulation of Membrane Transport

O-linked N-acetylglucosamine (O-GlcNAc) is a posttranslational modification that is increasingly recognized as a signal transduction mechanism. Unlike other glycans, O-GlcNAc is a highly dynamic and reversible process that involves the addition and removal of a single N-acetylglucosamine molecule to Ser/Thr residues of proteins. UDP-GlcNAc—the direct substrate for O-GlcNAc modification—is controlled by the rate of cellular metabolism, and thus O-GlcNAc is dependent on substrate availability. Serving as a feedback mechanism, O-GlcNAc influences the regulation of insulin signaling and glucose transport. Besides nutrient sensing, O-GlcNAc was also implicated in the regulation of various physiological and pathophysiological processes. Due to improvements of mass spectrometry techniques, more than one thousand proteins were detected to carry the O-GlcNAc moiety; many of them are known to participate in the regulation of metabolites, ions, or protein transport across biological membranes. Recent studies also indicated that O-GlcNAc is involved in stress adaptation; overwhelming evidences suggest that O-GlcNAc levels increase upon stress. O-GlcNAc elevation is generally considered to be beneficial during stress, although the exact nature of its protective effect is not understood. In this review, we summarize the current data regarding the oxidative stress-related changes of O-GlcNAc levels and discuss the implications related to membrane trafficking

Elevated Vascular Level of ortho-Tyrosine Contributes to the Impairment of Insulin-Induced Arterial Relaxation.

Previous studies have shown that in diabetes mellitus, insulin-induced relaxation of arteries is impaired and the level of ortho-tyrosine (o-Tyr), an oxidized amino acid is increased. Thus, we hypothesized that elevated vascular level of o-Tyr contributes to the impairment of insulin-induced vascular relaxation. Rats were fed with o-Tyr for 4 weeks. Insulin-induced vasomotor responses of isolated femoral artery were studied using wire myography. Vascular o-Tyr content was measured by HPLC, whereas immunoblot analyses were preformed to detect eNOS phosphorylation. Sustained oral supplementation of rats with o-Tyr increased the content of o-Tyr in the arterial wall and significantly reduced the relaxations to insulin. Sustained supplementation of cultured endothelial cells with o-Tyr increased the incorporation of o-Tyr and mitigated eNOS Ser (1 177) phosphorylation to insulin. Increasing arterial wall o-Tyr level attenuates insulin-induced relaxation - at least in part - by decreasing eNOS activation. Elevated level of o-Tyr could be an underlying mechanism for vasomotor dysfunction in diabetes mellitus

O-Linked N-Acetylglucosamine Transiently Elevates in HeLa Cells during Mitosis

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification of serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc modification influences many cellular mechanisms, including carbohydrate metabolism, signal transduction and protein degradation. Multiple studies also showed that cell cycle might be modulated by O-GlcNAc. Although the role of O-GlcNAc in the regulation of some cell cycle processes such as mitotic spindle organization or histone phosphorylation is well established, the general behaviour of O-GlcNAc regulation during cell cycle is still controversial. In this study, we analysed the dynamic changes of overall O-GlcNAc levels in HeLa cells using double thymidine block. O-GlcNAc levels in G1, S, G2 and M phase were measured. We observed that O-GlcNAc levels are significantly increased during mitosis in comparison to the other cell cycle phases. However, this change could only be detected when mitotic cells were enriched by harvesting round shaped cells from the G2/M fraction of the synchronized cells. Our data verify that O-GlcNAc is elevated during mitosis, but also emphasize that O-GlcNAc levels can significantly change in a short period of time. Thus, selection and collection of cells at specific cell-cycle checkpoints is a challenging, but necessary requirement for O-GlcNAc studies

A paclitaxel citotoxikus hatásának és a fehérjék o-glikozilációjának sejtciklusfüggő változásai

A sejtciklus zavarainak jelentősége számos betegség pathogenezisében került leírásra az elmúlt évek során, így a sejtciklusfüggő változások ismerete diagnosztikai és terápiás megközelítésből is igen lényegessé vált. A terápiás hatás fokozása volt célunk a dolgozat alapját képező munka első felében, mely során egy napjainkban is alkalmazott kemoterápiás szer, a paclitaxel sejtciklusfüggő effektivitásbeli különbségeit vizsgáltuk. A paclitaxel a sejtek szinkronizációját okozza a mitózis (M) fázisban. Vizsgálatainkkal arra kerestük a választ, hogy fokozható-e a szer citotoxikus hatása, amennyiben a kezelni kívánt sejtvonal osztódási karakterisztikáját figyelembe véve időzített, ismétlődő kezeléseket alkalmazunk. Egy gyorsan szaporodó sejtvonal (Sp2) osztódási ciklusára alapozott kezelési séma kidolgozását követően kimutattuk, hogy a második kezelés hatékonysága a G2/M fázisban a legmagasabb. Egy hosszabb duplikációs (generációs) idővel bíró sejttípussal (Jurkat) létrehozott kevert tenyészetet az Sp2-re optimalizált kezeléseknek kitéve képesek voltunk a két sejtvonalon eltérő hatás kifejtésére. Eredményeink azt mutatják, hogy az osztódási karakterisztika ismeretében a kemoterápiás hatékonyság növelhető. Megfelelően időzített kezelésekkel az egészséges testi sejteken okozott mellékhatás csökkenése mellett a tumorsejtekre kifejtett citotoxicitás fokozható. A munka második részében a sejtciklus-szabályozásban szerepet játszó egyik poszttranszlációs módosulás, az O-típusú fehérjeglikoziláció (O-GlcNAc) változásait követtük végig a sejtciklus során, szinkronizált HeLa sejteken. Bár a módosulásnak a sejtciklus bizonyos mozzanataiban betöltött szerepe részletesen vizsgált, a globális Oglikozilációs státusz osztódás alatti változásairól szóló közlemények ellentmondásosak. Munkánk során kimutattuk, hogy a mitózis folyamán az O-GlcNAc szint jelentősen megemelkedik, western blottal ugyanekkor egy jellegzetes O-GlcNAc-mintázatbeli eltérést azonosítható, egy 100 kDa magasságban megjelenő jel formájában. A malignus sejtek magas tápanyagfelvételének és emelkedett O-glikozilációs státuszának ismerete mellett eredményünk, miszerint a mitózis alatt a „nutrient sensor” funkciót betöltő OGlcNAc módosulás jelentősen megnő, tovább erősíti azt a korábbi feltételezést, hogy az O-glikoziláció és az azt reguláló enzimek a tumorképződés folyamatának fontos szereplői

O-Linked N-Acetylglucosamine Transiently Elevates in HeLa Cells during Mitosis

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification of serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc modification influences many cellular mechanisms, including carbohydrate metabolism, signal transduction and protein degradation. Multiple studies also showed that cell cycle might be modulated by O-GlcNAc. Although the role of O-GlcNAc in the regulation of some cell cycle processes such as mitotic spindle organization or histone phosphorylation is well established, the general behaviour of O-GlcNAc regulation during cell cycle is still controversial. In this study, we analysed the dynamic changes of overall O-GlcNAc levels in HeLa cells using double thymidine block. O-GlcNAc levels in G1, S, G2 and M phase were measured. We observed that O-GlcNAc levels are significantly increased during mitosis in comparison to the other cell cycle phases. However, this change could only be detected when mitotic cells were enriched by harvesting round shaped cells from the G2/M fraction of the synchronized cells. Our data verify that O-GlcNAc is elevated during mitosis, but also emphasize that O-GlcNAc levels can significantly change in a short period of time. Thus, selection and collection of cells at specific cell-cycle checkpoints is a challenging, but necessary requirement for O-GlcNAc studies

Protein O-GlcNAc Modification Increases in White Blood Cells After a Single Bout of Physical Exercise

BackgroundProtein O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification influencing the function of many intracellular proteins. Recently it was revealed that O-GlcNAc regulation is modified under various stress states, including ischemia and oxidative stress. Aside from a few contradictory studies based on animal models, the effect of exercise on O-GlcNAc is unexplored.PurposeTo evaluate O-GlcNAc levels in white blood cells (WBC) of human volunteers following physical exercise.MethodsYoung (age 30 ± 5.2), healthy male volunteers (n = 6) were enlisted for the study. Blood parameters including metabolites, ions, “necro”-enzymes, and cell counts were measured before and after a single bout of exercise (2-mile run). From WBC samples, we performed western blots to detect O-GlcNAc modified proteins. The distribution of O-GlcNAc in WBC subpopulations was assessed by flow cytometry.ResultsElevation of serum lactic acid (increased from 1.3 ± 0.4 to 6.9 ± 1.7 mM), creatinine (from 77.5 ± 6.3 U/L to 102.2 ± 7.0 μM), and lactate dehydrogenase (from 318.5 ± 26.2 to 380.5 ± 33.2 U/L) confirmed the effect of exercise. WBC count also significantly increased (from 6.6 ± 1.0 to 8.4 ± 1.4 G/L). The level of O-GlcNAc modified proteins in WBCs showed significant elevation after exercise (85 ± 51%, p < 0.05). Flow cytometry revealed that most of this change could be attributed to lymphocytes and monocytes.ConclusionOur results indicate that short-term exercise impacts the O-GlcNAc status of WBCs. O-GlcNAc modification could be a natural process by which physical activity modulates the immune system. Further research could elucidate the role of O-GlcNAc during exercise and validate O-GlcNAc as a biomarker for fitness assessment