Tissue transglutaminase modulates α‐synuclein oligomerization

Abstract

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's diseaseassociated a-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant a-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective a-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/a-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all a-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal a-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal b-sheetcontaining oligomers, the tTG/a-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant a-synuclein variants. We propose that tTG cross-linking imposes structural constraints on asynuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

“…As presented in Fig. 1D, the NMR spectra of ␣-syn obtained for the two different purification procedures are nearly identical, and both clearly indicate the highly disordered conformational ensemble that was originally documented (23) and has since been extensively studied by us (39 -46) and many other groups (47)(48)(49)(50)(51)(52)(53).…”

Transglutaminases and Disease: Lessons From Genetically Engineered Mouse Models and Inherited Disorders

“…235), mechanistic studies in TG2-modified animals, aimed at evaluating the role of TG2 in these diseases, have not yet been undertaken. Recent studies suggest that TG2-mediated intraand intermolecular cross-linked ␣-synuclein is unable to form the cytotoxic, structured fibrillar aggregates responsible for neural membrane disruption in Parkinson's disease (247). TG2-mediated cross-linking may, thus, prevent progression of Parkinson's disease.…”

Transglutaminase Activity as a Possible Molecular Mechanism in the Etiopathogenesis of Neurodegenerative Diseases

“…However, although all these studies suggest the possible involvement of the TGs in the formation of deposits of protein aggregates in neurodegenerative diseases, they do not indicate whether aberrant TG activity per se directly determines the disease progression. For example, several experimental findings reported that TG2 activity in vitro leads to the formation of soluble aggregates of a-synuclein [47] or polyQ proteins [48,49] . To date, as previously reported, at least ten human CAG-expansion diseases have been described (Table 2) [50][51][52][53][54][55][56][57][58][59] and in at least eight of them their neuropathology is caused by the expansion in the number of residues in the polyglutamine domain to a value beyond [35][36][37][38][39][40].…”