Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions

2009 ◽  
Vol 423 (2) ◽  
pp. 253-264 ◽  
Author(s):  
Na Rae Hwang ◽  
Seung-Hee Yim ◽  
Young Mee Kim ◽  
Jaeho Jeong ◽  
Eun Joo Song ◽  
...  

Knowledge of the cellular targets of ROS (reactive oxygen species) and their regulation is an essential prerequisite for understanding ROS-mediated signalling. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is known as a major target protein in oxidative stresses and becomes thiolated in its active site. However, the molecular and functional changes of oxidized GAPDH, the inactive form, have not yet been characterized. To examine the modifications of GAPDH under oxidative stress, we separated the oxidation products by two-dimensional gel electrophoresis and identified them using nanoLC-ESI-q-TOF MS/MS (nano column liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem MS). Intracellular GAPDH subjected to oxidative stress separated into multiple acidic spots on two-dimensional gel electrophoresis and were identified as cysteine disulfide and cysteic acids on Cys152 in the active site. We identified the interacting proteins of oxidized inactive GAPDH as p54nrb (54 kDa nuclear RNA-binding protein) and PSF (polypyrimidine tract-binding protein-associated splicing factor), both of which are known to exist as heterodimers and bind to RNA and DNA. Interaction between oxidized GAPDH and p54nrb was abolished upon expression of the GAPDH active site mutant C152S. The C-terminal of p54nrb binds to GAPDH in the cytosol in a manner dependent on the dose of hydrogen peroxide. The GAPDH–p54nrb complex enhances the intrinsic topoisomerase I activation by p54nrb–PSF binding. These results suggest that GAPDH exerts other functions beyond glycolysis, and that oxidatively modified GAPDH regulates its cellular functions by changing its interacting proteins, i.e. the RNA splicing by interacting with the p54nrb–PSF complex.

1985 ◽  
Vol 5 (3) ◽  
pp. 586-590
Author(s):  
A M Francoeur ◽  
E K Chan ◽  
J I Garrels ◽  
M B Mathews

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.


2013 ◽  
Vol 451 (2) ◽  
pp. 277-288 ◽  
Author(s):  
Sarah Schlosser ◽  
David Leitsch ◽  
Michael Duchêne

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.


2008 ◽  
Vol 2008 ◽  
pp. 1-6 ◽  
Author(s):  
Xuesong Sun ◽  
Ruiguang Ge ◽  
Jen-Fu Chiu ◽  
Hongzhe Sun ◽  
Qing-Yu He

Helicobacter pylori (H. pylori) is a widespread human pathogen causing peptic ulcers and chronic gastritis. Maintaining nickel homeostasis is crucial for the establishment of H. pylori infection in humans. We used immobilized-nickel affinity chromatography to isolate Ni-related proteins from H. pylori cell extracts. Two-dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two Ni-interacting proteins in H. pylori. These Ni-interacting proteins can be classified into several general functional categories, including cellular processes (HspA, HspB, TsaA, and NapA), enzymes (Urease, Fumarase, GuaB, Cad, PPase, and DmpI), membrane-associated proteins (OM jhp1427 and HpaA), iron storage protein (Pfr), and hypothetical proteins (HP0271, HP jhp0216, HP jhp0301, HP0721, HP0614, and HP jhp0118). The implication of these proteins in nickel homeostasis is discussed.


1986 ◽  
Vol 240 (2) ◽  
pp. 593-596
Author(s):  
P Strocchi ◽  
J M Gilbert

One of the most abundant acidic proteins in rat brain has an Mr of 68,000 and a pI of 5.6 (68K 5.6 protein) when analysed by two-dimensional gel electrophoresis. The 68K 5.6 protein was found in large relative amounts in brain cytoskeleton preparations and in membrane and supernatant fractions. High-salt washing and proteolytic digestion did not remove this protein from the membrane elements. The 68K 5.6 protein was also found in the microtubule-associated protein fraction of purified microtubules and was present in large relative amounts in preparations of intermediate-filament proteins. The 68K 5.6 protein binds to calmodulin in the presence of Ca2+ ions, and we found it to be an abundant acidic calmodulin-binding protein in brain tissue.


1985 ◽  
Vol 5 (3) ◽  
pp. 586-590 ◽  
Author(s):  
A M Francoeur ◽  
E K Chan ◽  
J I Garrels ◽  
M B Mathews

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.


1985 ◽  
Vol 54 (03) ◽  
pp. 626-629 ◽  
Author(s):  
M Meyer ◽  
F H Herrmann

SummaryThe platelet proteins of 9 thrombasthenic patients from 7 families were analysed by high resolution two-dimensional gel electrophoresis (HR-2DE) and crossed immunoelectrophoresis (CIE). In 7 patients both glycoproteins (GPs) IIb and Ilia were absent or reduced to roughly the same extent. In two related patients only a trace of GP Ilb-IIIa complex was detected in CIE, but HR-2DE revealed a glycopeptide in the position of GP Ilia in an amount comparable to type II thrombasthenia. This GP Ilia-like component was neither recognized normally by anti-GP Ilb-IIIa antibodies nor labeled by surface iodination. In unreduced-reduced two-dimensional gel electrophoresis two components were observed in the region of GP Ilia. The assumption of a structural variant of GP Ilia in the two related patients is discussed.


2015 ◽  
Vol 22 (12) ◽  
pp. 1066-1075 ◽  
Author(s):  
Adriana Magalhães ◽  
Rayner Queiroz ◽  
Izabela Bastos ◽  
Jaime Santana ◽  
Marcelo Sousa ◽  
...  

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