Dissecting the mechanisms of tissue transglutaminase-induced cross-linking of alpha-synuclein: implications for the pathogenesis of Parkinson disease

Diego Chiappe, Ivan Hang, Hilal Lashuel, Marc Moniatte, Adrian Schmid
American Society for Biochemistry and Molecular Biology, 2009
Journal paper

Abstract

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of alpha-synuclein (alpha-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type alpha-syn and alpha-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of alpha-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric alpha-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric alpha-syn composed of either wild-type or Gln --> Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of alpha-syn. These studies demonstrate for the first time that Gln(79) and Gln(109) serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of alpha-syn and tTG-induced inhibition of alpha-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on alpha-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of alpha-syn.

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Diego Chiappe, Ivan Hang, Hilal Lashuel, Marc Moniatte, Adrian Schmid
American Society for Biochemistry and Molecular Biology, 2009
Journal paper

Abstract

Official source

https://infoscience.epfl.ch/record/142096?ln=en

About this result

Related concepts (15)

Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass

Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mu

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Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease share common mechanisms characterized by protein misfolding and aggregation, including formation of plaques or inclusion bodies. The rapid growing number of patients has motivated intensive research to understand biological phenomena involved in the pathology and hence developing therapeutic strategies. Here, we will focus on β-amyloid peptide (Aβ) involved in Alzheimer's disease. In the first part of this thesis, tissue transglutaminase (tTGase) has been investigated due to its implication in a number of cellular processes and disease states, where the enzymatic actions of tTGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of tTGase in cell survival or cell death mechanisms still remain elusive. tTGase mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that, tTGase induces intramolecular cross-linking of Aβ peptide, resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that, a large population of Aβ monomers contained an increase in mass of 0.984 Da at a glutamine residue, indicating that glutamine Q15 serves as an indispensable substrate in TGase mediated deamidation to glutamate E15. We provide strong analytical evidence on tTGase mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that, tTGase induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation. In the second part of this work, we explore the idea of using synthetic peptides that can switch conformation in a controlled way as a sensor for β-breakers of Aβ. The concept of switch-peptides allows the controlled onset of polypeptides folding and misfolding at physiological pH by in situ O,N-acyl migration. The synthesized sequence consisted in the core sequence responsible for Aβ aggregation, flanked by two β-sheet-inducing template through ester bonds. The triggering of the acyl migration allows the native sequence to be recovered from the ester bonds and structural change from flexible unordered conformation to β-sheets and subsequently fibrils formation were observed in solution. In a more biological environment made of lipid vesicles, the conformation of the peptide has been shown to act comparably to wild type Aβ peptide by circular dichroism (CD), namely increased negative charges over the surface led to increased helical propensity followed by a transition to β-sheets and accelerated fibrils formation. Thereafter, the conformation of the cysteine analogue of the switch-peptide covalently linked to gold surface was characterized by Fourier-transform infrared spectroscopy (FTIR). In monolayer of switch-peptide tethered to a planar gold surface, the peptide already adopts most likely β-turn conformation and no changes in the infrared spectra were detected after the triggering of the switch. On gold nanoparticles, the switch-peptide folds spontaneously into β-sheet conformation. These unwanted foldings limit the efficiency of the sensor and has driven us to an alternative configuration, where gold nanoparticles were coated with a 95:5 mixture of two peptide sequences able to stabilize the nanoparticles and to act as recognition sequence, respectively. In this configuration, the switch-peptide is added in the gold nanoparticles solution and the aggregation triggered. Here, we want to take advantage of a particular feature of gold nanoparticles that consists in a change of solution color from red to blue as the nanoparticles come closer to each other during aggregation. Preliminary results have shown that a discrimination between aggregated and soluble samples is possible. However, further improvements are necessary before a potential biosensor could be expected.

EPFL2011

Phage Selection of Bicyclic Peptide Ligands and Development of a New Peptide Cyclisation Method

Jeremy Touati

Bicyclic peptides binding to targets of interest can be isolated from combinatorial libraries using a phage display-based approach. In a first project of this thesis, we aimed at generating bicyclic peptide inhibitors of matrix metalloproteinases (MMPs), a family of proteases that share high structural similarities and have been difficult to target in a specific manner. From phage-encoded combinatorial libraries, we isolated a bicyclic peptide that inhibits specifically the gelatinase MMP-2 with a Ki of 2 μM. This inhibitor was less potent than the best peptidic MMP-2 inhibitor, the linear APP-derived inhibitory peptide (Ki = 13 nM). However, due to its conformational constraint, the bicyclic peptide is expected to be significantly more stable and hence more suitable for applications in biological systems. It may be used as a research tool alternatively to the broadly applied monocyclic peptide MMP-2 inhibitor CTT which is less potent (Ki of 142 μM). If affinity matured, the bicyclic peptide MMP-2 inhibitor might even be developed into an anti-cancer therapeutic. In a second project, bicyclic peptide binders to the centriolar protein SAS-6 were generated. SAS-6 plays an important structural role in centrioles and bicyclic peptide binders are currently applied in cellular assays by the laboratory of Professor Pierre Gönczy (EPFL) to study the process of centriole formation. In a third project of this thesis, we established an enzymatic method to selectively cyclise peptides with unprotected side chains using a transglutaminase (TGase). TGases catalyse the formation of stable amide bonds between the side chains of glutamine and primary amines. They have been used extensively in biotechnological applications to cross-link peptides and proteins or to attach labels to proteins. We found that the microbial transglutaminase (MTGase) of Streptomyces mobaraensis can cyclise quantitatively peptides with an N- terminal glutamine-donor sequence and a C-terminal lysine residue in an intramolecular reaction. Experiments with minimised glutamine-donor substrates revealed that peptides with only an Ala-Leu dipeptide N-terminal to the glutamine and a randomly chosen peptide between the glutamine and lysine residues are efficiently cyclised. By combining the MTGase-based cyclisation strategy with a chemical thiol-based cyclisation reaction, we were able to generate tricyclic peptide structures. It remains to be tested if this method can be applied to cyclise combinatorial peptide libraries on the surface of phage. In a fourth and last project, we developed a method to follow qualitatively and quantitatively chemical or enzymatic modifications applied to peptides displayed on phage. In this method, peptides on phage were chemically modified, cleaved off by a protease, purified, concentrated and analysed by mass spectrometry. The method will help to assess new reactions applied to phage peptides such as the MTGase-based enzymatic cyclisation reaction.

EPFL2012

Cyclic peptides have a range of properties that make them attractive as therapeutics such as high binding affinity, good target selectivity and low toxicity. While most peptides in the clinic are derived from nature, various powerful in vitro evolution techniques allow now the de novo development of cyclic peptide ligands to targets of interest. Our group is specialized in the development of bicyclic peptide ligands by phage display. A current limitation of phage-selected bicyclic peptides is their conformational flexibility limiting the binding affinity due to entropic effects or even preventing the generation of binders to challenging targets. The main goal of my thesis was the development of new bicyclic peptide formats that are conformationally more rigid or even have a stable fold in solution. The proposed bicyclic peptide formats are based on a rigid chemical structure containing multiple polar groups and peptides that are anchored to the structure and fold tightly around by forming non-covalent interactions. I also envisioned to cyclise peptide phage display libraries with several such chemical structures in parallel to generate and screen structurally highly diverse bicyclic peptide libraries. In a first project, I designed and synthesized three new compounds that have i) three thiol-reactive groups to react with peptides and ii) several polar groups. Two of these compounds efficiently and selectively cyclised linear peptides containing three cysteine residues. Functional and structural analysis of the bicyclic peptides showed that the relatively small compounds at the centre of bicyclic peptides significantly influence the activity and conformation of the peptides. These results demonstrated the prominent structural role of chemical linkers in peptide macrocycles. In a second project, I applied the compounds developed in the first project in phage selections. The sequences of isolted bicyclic peptide ligands were strongly influenced by the small chemical structures, suggesting that the peptides fold closely around them. X-ray structure analysis revealed that one of the chemical structures indeed nucleated peptides by forming H-bond interactions. This result showed that small rigid chemicals can serve as nucleating scaffolds and directs the way towards the generation of peptide ligands being folded in solution. A third project is based on an unexpected observation in the second project, namely that panning peptide phage libraries without prior alkylation with compounds yielded mostly peptides with four cysteines. The peptides bound with good affinity and characterization revealed that they contain two disulphide bridges and thus formed bicyclic peptide structures. An interesting feature of disulfide-based bicyclic peptide libraries was the high topological diversity: oxidation of peptide libraries of the form Xm CXn CXo CXp yielded 3 × (m+n+o+p) different bicyclic peptide topologies because the fourth cysteine could appear in any of the (m+n+o+p) randomized amino acid positions (X) and the four peptides in such peptides could pair in three different ways. X-ray structure analysis revealed an important structural role of the disulfide bridges. The new method offers an easy and robust way to generate bicyclic peptide ligands.

EPFL2013

Phage Selection of Bicyclic Peptide Ligands and Development of a New Peptide Cyclisation Method

Jeremy Touati

EPFL2012

EPFL2013

EPFL2011