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Structural characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase protofibrils preventing ?-synuclein oligomeric species toxicity.

ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with ?-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of ?-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and ?-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by ?-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping ?-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

SUBMITTER: Avila CL

PROVIDER: S-EPMC4022857 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Structural characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase protofibrils preventing α-synuclein oligomeric species toxicity.

Ávila César L CL Torres-Bugeau Clarisa M CM Barbosa Leandro R S LR Sales Elisa Morandé EM Ouidja Mohand O MO Socías Sergio B SB Celej M Soledad MS Raisman-Vozari Rita R Papy-Garcia Dulce D Itri Rosangela R Chehín Rosana N RN

The Journal of biological chemistry 20140326 20

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be de ...[more]

PMID: 24671416

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Project description:Study hypothesisDetailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development.Study findingThis study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility.What is known alreadyGlycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function.Study design, samples/materials, methodsHomology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell.Main results and the role of chanceOur studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 μM compared with an IC50 of 38.5 μM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 μM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions).Limitations, reasons for cautionThe chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development.Wider implications of the findingsThis study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets.Large scale dataNone. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org).Study funding and competing interestsThis work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.

Dataset Information

Structural characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase protofibrils preventing ?-synuclein oligomeric species toxicity.

Publications

Structural characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase protofibrils preventing α-synuclein oligomeric species toxicity.

Similar Datasets

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