9 research outputs found
Transglutaminase and diseases of the central nervous system.
Alzheimer's disease, Parkinson's disease and diseases of expanded polyglutamine are associated with insoluble protein aggregates and neuronal death. A role for transglutaminase in the stabilization of these aggregates has been proposed. Diseases of polyglutamine expansion have been the most thoroughly investigated and a large body of studies supports the causative role of transglutaminase in aggregation of expanded polyglutamine. However none of the evidence is conclusive. Indisputable evidence of cross-linking by transglutaminase will be required in order to provide firm support for therapeutic measures based on the role of transglutaminase
Purification of neuronal inclusions of patients with Huntington disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers
Purification of neuronal inclusions of patients with Huntington disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers
[N(epsilon)-(gamma-glutamyl) lysine] as a potential biomarker in neurological diseases : New detection method and fragmentation pathways.
Protein aggregates are characteristic of a number of diseases of the central nervous system such as diseases of polyQ expansion. Covalent bonds formed by the action of transglutaminase are thought to participate in the stabilization of these aggregates. Transglutaminase catalyzes the formation of cross-links between the side chains of glutaminyl and lysyl residues of polypeptides. Identification of the isodipeptide N(epsilon)-(gamma-glutamyl) lysine (iEK) in terminal proteolytic digests of neuronal aggregates would demonstrate participation of transglutaminase in neurological diseases. In order to identify and quantify the iEK present in the brain of patients with neurological disease, a method combining liquid chromatography and multistep mass spectrometry was developed. Because isobaric peptides of iEK could be present in the digest of aggregated proteins, the choice of fragment diagnostic ions was crucial. These ions were identified by mass spectrometry on sodiated iEK, which was derivatized on the carboxylic functions and terminal amines in order to improve sensitivity. Deuterated molecules as well as (13)C(6)- and (15)N(2)-isotopomers were used to derive filiations in the multistep fragmentations. The main fragmentation patterns have been identified, so that two ions (m/z 396 [MH - 56-42 u](+) and 350 [MH - 56-88 u](+)) are shown to be adequate markers for quantitation experiments. In order to gain a better understanding of the fragmentation processes, detailed quantum chemical calculations have been performed at levels which are expected to provide good accuracy. A thorough study has been carried out with a reduced model in which only the 'active' part of the molecule is retained. This allowed obtaining full mechanistic details on the pathways leading to a number of observed fragments. In particular, it has been shown that losses of 87 and 88 u from A(+) = [MH - 56 u](+) are competitive. Computations on the entire derivatized isodipeptide have been used to validate the use of the smaller model in order to obtain reliable energetics and mechanisms. Copyright (c) 2007 John Wiley & Sons, Ltd
Inclusions of R6/2 Mice Are Not Amyloid and Differ Structurally from Those of Huntington Disease Brain
R6/2
mice contain an N-terminal fragment of human huntingtin with
an expanded polyQ and develop a neurological disease resembling Huntington
disease. Although the brain of R6/2 mice contains numerous inclusions,
there is very little neuronal death. In that respect, R6/2 mice differ
from patients with Huntington disease whose striatum and cerebral
cortex develop inclusions associated with extensive neuronal loss.
We have previously demonstrated using synchrotron-based infrared microspectroscopy
that the striatum and the cortex of patients with Huntington disease
contained inclusions specifically enriched in amyloid ÎČ-sheets.
We had concluded that the presence of an amyloid motif conferred toxicity
to the inclusions. We demonstrate here by synchrotron based infrared
microspectroscopy in transmission and attenuated total reflectance
mode that the inclusions of R6/2 mice possess no detectable amyloid
and are composed of proteins whose structure is not distinguishable
from that of the surrounding soluble proteins. The difference in structure
between the inclusions of patients affected by Huntington disease
and those of R6/2 mice might explain why the former but not the latter
cause neuronal death
Structure of Inclusions of Huntingtonâs Disease Brain Revealed by Synchrotron Infrared Microspectroscopy: Polymorphism and Relevance to Cytotoxicity
Huntingtonâs disease is caused
by a polyglutamine expansion
in huntingtin. Affected brain regions contain characteristic aggregates
of the misfolded expanded protein. Studies in cells and animals show
that aggregates are polymorphic and that the secondary structure of
the aggregates is likely to condition their cytotoxicity. Therefore
knowing the structure of aggregates is important as neurotoxic secondary
structures may be specifically targeted during the search for prophylactic
or therapeutic drugs. The structure of aggregates in the brain of
patients is still unknown. Using synchrotron based infrared microspectroscopy
we demonstrate that the brains of patients with Huntington disease
contain putative oligomers and various kinds of microscopic aggregates
(inclusions) that can be distinguished by their differential absorbance
at 1627 cm<sup>â1</sup> (amyloid ÎČ sheets) and 1639 cm<sup>â1</sup> (ÎČ sheets/unordered). We also describe the
parallel/antiparallel organization of the ÎČ strands. As the
inclusions enriched in both ÎČ sheets and ÎČ sheets/unordered
structures are characteristic of severely affected brain regions,
we conclude that this kind of amyloid inclusions is likely to be particularly
toxic to neurons
Misfolding of Proteins with a Polyglutamine Expansion Is Facilitated by Proteasomal Chaperones*Sâ
Deposition of misfolded proteins with a polyglutamine expansion is a
hallmark of Huntington disease and other neurodegenerative disorders.
Impairment of the proteolytic function of the proteasome has been reported to
be both a cause and a consequence of polyglutamine accumulation. Here we found
that the proteasomal chaperones that unfold proteins to be degraded by the
proteasome but also have non-proteolytic functions co-localized with
huntingtin inclusions both in primary neurons and in Huntington disease
patients and formed a complex independently of the proteolytic particle.
Overexpression of Rpt4 or Rpt6 facilitated aggregation of mutant huntingtin
and ataxin-3 without affecting proteasomal degradation. Conversely, reducing
Rpt6 or Rpt4 levels decreased the number of inclusions in primary neurons,
indicating that endogenous Rpt4 and Rpt6 facilitate inclusion formation.
In vitro reconstitution experiments revealed that purified 19S
particles promote mutant huntingtin aggregation. When fused to the ornithine
decarboxylase destabilizing sequence, proteins with expanded polyglutamine
were efficiently degraded and did not aggregate. We propose that aggregation
of proteins with expanded polyglutamine is not a consequence of a proteolytic
failure of the 20S proteasome. Rather, aggregation is elicited by chaperone
subunits of the 19S particle independently of proteolysis