Dynamic Oligomeric Properties

Seidler, Norbert W.

doi:10.1007/978-94-007-4716-6_7

Cited by 5 publications

(3 citation statements)

References 136 publications

(196 reference statements)

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“…7A). The R axis marks the dimerdimer interface; the P axis is the interface with the most contacts between two monomeric subunits, and these contacts are highly conserved in evolution (38,39). Relevant to our study, we found that the sequence from which the GAPDH peptide was derived as well as the phosphorylation site Thr-246 are both found on the P axis (Fig.…”

Section: Rational Design Of a Peptide Based On Homology Betweensupporting

confidence: 80%

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death

Qvit

Joshi

Cunningham

et al. 2016

Journal of Biological Chemistry

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a noncanonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by ␦ protein kinase C (␦PKC) inhibits this GAPDH-dependent mitochondrial elimination. ␦PKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudo-GAPDH (GAPDH) peptide, an inhibitor of ␦PKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other ␦PKC substrates. Unexpectedly, GAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with GAPDH or direct phosphorylation of GAPDH by ␦PKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo. Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDHmediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, GAPDH peptide, with interesting properties. GAPDH peptide is an inhibitor of the interaction between ␦PKC and GAPDH and of the resulting phosphorylation of GAPDH by ␦PKC. GAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that GAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for GAPDH peptide-based therapy.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. We previously found that in addition to its role as a glycolytic enzyme, GAPDH is also involved in mitochondrial elimination by mitophagy (1). GAPDH binds to damaged mitochondria, and its phosphorylation by ␦ protein kinase C (␦PKC) under oxidative stress inhibits the removal of the damaged mitochondria through mitophagy (1). We set out to determine whether ␦PKC-mediated GAPDH phosphorylation alone is sufficient to mediate cell death following oxidative stress, using Langendorff, an ex vivo model of heart attack.We first designed a novel inhibitor that selectively inhibits GAPDH phosphorylation without affecting the phosphorylation of other ␦PKC substrates. To this end, we relied on the rationale that, similar to many other protein kinases, ␦PKC contains a highly conserved catalytic domain and a regulatory domain that keeps the enzyme in an inactive conformation. This inactive conformation is stabilized by several intramolecular auto-inhibitory interactions (2). The first intramolecular interaction identified was the one mediated by the pseudosubstrate site located at the N terminus of the kinase, which mimics the phospho-acceptor sequence in PKC substrate...

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Section: Rational Design Of a Peptide Based On Homology Betweensupporting

confidence: 80%

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death

Qvit

Joshi

Cunningham

et al. 2016

Journal of Biological Chemistry

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“…A total of 13 AMP-PNP-induced stabilizations were detected in this work ( Table 2). The K d values calculated from these ligandinduced stabilizations ranged from <100 μM to 2.0 mM, which is in the range of previously reported ATP binding affinities [41][42][43].…”

Section: Atp-induced Stabilization Versus Destabilizationmentioning

confidence: 60%

Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

Geer

Fitzgerald

2015

J. Am. Soc. Mass Spectrom.

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Abstract. The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATPbinding proteins in the Saccharomyces Genome Database (SGD). The 14 nonannotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

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“…Protein oligomerization has functional implications in a variety of biological processes (27,28,29). The full-length RspWYL1 protein eluted at a volume equivalent to a molecular weight of ∼90 kDa on a Superdex 200 gel filtration column, matching the theoretical molecular weight of dimeric RspWYL1, suggesting that RspWYL1 exists as a dimer in solution (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

Structural insights into the modulatory role of the accessory protein WYL1 in the Type VI-D CRISPR-Cas system

Cheng

Wasney

et al. 2019

Nucleic Acids Research

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The Type VI-D CRISPR-Cas system employs an RNA-guided RNase Cas13d with minimal targeting constraints to combat viral infections. This CRISPR system contains RspWYL1 as a unique accessory protein that plays a key role in boosting its effector function on target RNAs, but the mechanism behind this RspWYL1-mediated stimulation remains completely unexplored. Through structural and biophysical approaches, we reveal that the full-length RspWYL1 possesses a novel three-domain architecture and preferentially binds ssRNA with high affinity. Specifically, the N-terminus of RspWYL1 harbors a ribbon-helix-helix motif reminiscent of transcriptional regulators; the central WYL domain of RspWYL1 displays a Sm-like β-barrel fold; and the C-terminal domain of RspWYL1 primarily contributes to the dimerization of RspWYL1 and may regulate the RspWYL1 function via a large conformational change. Collectively, this study provides a first glimpse into the complex mechanism behind the RspWYL1-dictated boosting of target ssRNA cleavage in the Type VI-D CRISPR-Cas system.

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Dynamic Oligomeric Properties

Cited by 5 publications

References 136 publications

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death

Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

Structural insights into the modulatory role of the accessory protein WYL1 in the Type VI-D CRISPR-Cas system

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