SUMO-1 regulates the conformational dynamics of Thymine-DNA Glycosylase regulatory domain and competes with its DNA binding activity

<p>Abstract</p> <p>Background</p> <p>The human thymine-DNA glycosylase (TDG) plays a dual role in base excision repair of G:U/T mismatches and in transcription. Regulation of TDG activity by SUMO-1 conjugation was shown to act on both functions. Furthermore, TDG can interact with SUMO-1 in a non-covalent manner.</p> <p>Results</p> <p>Using NMR spectroscopy we have determined distinct conformational changes in TDG upon either covalent sumoylation on lysine 330 or intermolecular SUMO-1 binding through a unique SUMO-binding motif (SBM) localized in the C-terminal region of TDG. The non-covalent SUMO-1 binding induces a conformational change of the TDG amino-terminal regulatory domain (RD). Such conformational dynamics do not exist with covalent SUMO-1 attachment and could potentially play a broader role in the regulation of TDG functions for instance during transcription. Both covalent and non-covalent processes activate TDG G:U repair similarly. Surprisingly, despite a dissociation of the SBM/SUMO-1 complex in presence of a DNA substrate, SUMO-1 preserves its ability to stimulate TDG activity indicating that the non-covalent interactions are not directly involved in the regulation of TDG activity. SUMO-1 instead acts, as demonstrated here, indirectly by competing with the regulatory domain of TDG for DNA binding.</p> <p>Conclusions</p> <p>SUMO-1 increases the enzymatic turnover of TDG by overcoming the product-inhibition of TDG on apurinic sites. The mechanism involves a competitive DNA binding activity of SUMO-1 towards the regulatory domain of TDG. This mechanism might be a general feature of SUMO-1 regulation of other DNA-bound factors such as transcription regulatory proteins.</p

Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions.

The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites-the global pattern of phosphorylation or 'phospho-form'-giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2(n) phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods-western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)-on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem

SUMO-1 possesses DNA binding activity

<p>Abstract</p> <p>Background</p> <p>Conjugation of small ubiquitin-related modifiers (SUMOs) is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs). Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood.</p> <p>Findings</p> <p>Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM.</p> <p>Conclusion</p> <p>This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.</p

Molecular Implication of PP2A and Pin1 in the Alzheimer's Disease Specific Hyperphosphorylation of Tau

Tau phosphorylation and dephosphorylation regulate in a poorly understood manner its physiological role of microtubule stabilization, and equally its integration in Alzheimer disease (AD) related fibrils. A specific phospho-pattern will result from the balance between kinases and phosphatases. The heterotrimeric Protein Phosphatase type 2A encompassing regulatory subunit PR55/Bα (PP2A(T55α)) is a major Tau phosphatase in vivo, which contributes to its final phosphorylation state. We use NMR spectroscopy to determine the dephosphorylation rates of phospho-Tau by this major brain phosphatase, and present site-specific and kinetic data for the individual sites including the pS202/pT205 AT8 and pT231 AT180 phospho-epitopes.We demonstrate the importance of the PR55/Bα regulatory subunit of PP2A within this enzymatic process, and show that, unexpectedly, phosphorylation at the pT231 AT180 site negatively interferes with the dephosphorylation of the pS202/pT205 AT8 site. This inhibitory effect can be released by the phosphorylation dependent prolyl cis/trans isomerase Pin1. Because the stimulatory effect is lost with the dimeric PP2A core enzyme (PP2A(D)) or with a phospho-Tau T231A mutant, we propose that Pin1 regulates the interaction between the PR55/Bα subunit and the AT180 phospho-epitope on Tau.Our results show that phosphorylation of T231 (AT180) can negatively influence the dephosphorylation of the pS202/pT205 AT8 epitope, even without an altered PP2A pool. Thus, a priming dephosphorylation of pT231 AT180 is required for efficient PP2A(T55α)-mediated dephosphorylation of pS202/pT205 AT8. The sophisticated interplay between priming mechanisms reported for certain Tau kinases and the one described here for Tau phosphatase PP2A(T55α) may contribute to the hyperphosphorylation of Tau observed in AD neurons

Structure of three Kdn-containing oligosaccharide-alditols released from oviducal secretions of Ambystoma tigrinum: characterization of the carbohydrate sequence Fuc (α1–5) [Fuc (α1–4)] Kdn (α2—3/6)

AbstractOligosaccharide-alditols released by reductive β-elimination from the egg jelly coat of Ambystoma tigrinum were analyzed by 1H NMR spectroscopy. As observed for five other amphibian species, these carbohydrate chains are highly species-specific, and should support the species-specificity of gamete interaction. The carbohydrate chains of Ambystoma tigrinum are characterized by the presence of a new type of sequence: Fuc (α1–5) [(Fuc (α1–4)] Kdn (α2–3/6)

Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana

The 119-amino acid residue prolyl cis/trans isomerase from Arabidopsis thaliana (PIN1At) is similar to the catalytic domain of the human hPIN1. However, PIN1At lacks the N-terminal WW domain that appears to be essential for the hPIN1 function. Here, the solution structure of PIN1At was determined by three-dimensional nuclear magnetic resonance spectroscopy. The PIN1At fold could be superimposed on that of the catalytic domain of hPIN1 and had a 19 residue flexible loop located between strand beta1 and helix alpha1. The dynamical features of this beta1/alpha1-loop, which are characteristic for a region involved in protein-protein interactions, led to exchange broadening in the NMR spectra. When sodium sulfate salt was added to the protein sample, the beta1/alpha1 loop was stabilized and, hence, a complete backbone resonance assignment was obtained. Previously, with a phospho-Cdc25 peptide as substrate, PIN1At had been shown to catalyze the phosphoserine/phosphothreonine prolyl cis/trans isomerization specifically. To map the catalytic site of PIN1At, the phospho-Cdc25 peptide or sodium sulfate salt was added in excess to the protein and chemical shift changes in the backbone amide protons were monitored in the (1)H(N)-(15)N heteronuclear single quantum coherence spectrum. The peptide caused perturbations in the loops between helix alpha4 and strand beta3, between strands beta3 and beta4, in the alpha3 helix, and in the beta1/alpha1 loop. The amide groups of the residues Arg21 and Arg22 showed large chemical shift perturbations upon phospho-Cdc25 peptide or sulfate addition. We conclude that this basic cluster formed by Arg21 and Arg22, both located in the beta1/alpha1 loop, is homologous to that found in the hPIN1 crystal structure (Arg68 and Arg69), which also is involved in sulfate ion binding. We showed that the sulfate group competed for the interaction between PIN1At and the phospho-Cdc25 peptide. In the absence of the WW domain, three hydrophobic residues (Ile33, Ile34, and Leu35) located in the long flexible loop and specific for the plant PIN-type peptidyl prolyl cis/trans isomerases (PPIases) could be an additional interaction site in PIN1At. However, phospho-peptide addition did not affect the resonances of these residues significantly. Electrostatic potential calculations revealed a negatively charged area not found in hPIN1 on the PIN1At molecular surface, which corresponds to the surface shielded by the WW domain in hPIN1. Based on our experimental results and the molecular specificities of the PIN1At enzyme, functional implications of the lack of WW domains in this plant PIN-type PPIase will be discussed.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Towards understanding the phosphorylation code of tau

Abstract We describe our efforts to combine in vitro enzymatic reactions with recombinant kinases to phosphorylate the neuronal tau protein, and NMR spectroscopy to unravel the resulting phosphorylation pattern in both qualitative and quantitative manners. This approach, followed by functional assays with the same samples, gives access to the complex phosphorylation code of tau. As a result, we propose a novel hypothesis for the link between tau (hyper)phosphorylation and aggregation