Lausanne: EPFL scientists have developed a new method that can
accurately simulate the chemical modification of the protein behind
Parkinson’s disease. The technique, has opened a new way of
understanding Parkinson’s, and can be expanded to other proteins and
diseases as well.
Parkinson’s disease is characterized by the
aggregation of the protein alpha-synuclein in brain cells that control
movement, giving rise to the disease’s symptoms. Evidence suggests that
alpha-synuclein begins to aggregate when it undergoes a chemical process
where nitrogen groups are attached to four of its amino acids. This
process, called nitration, has been impossible to study on
alpha-synuclein in the lab. Scientists at École Polytechnique Fédérale
de Lausanne (EPFL) have developed the first-ever method where
alpha-synuclein can be nitrated to simulate the nitration patterns seen
in Parkinson’s disease. The method, which can be expanded to investigate
the effect of nitration in other proteins, is published in the Journal of the American Chemical Society and opens new possibilities for understanding the role of this nitration in health and disease.
The
exact causes of Parkinson’s disease, which affects around ten million
people worldwide, are still unclear. But one place to look is the
protein alpha-synuclein, which begins to clump together inside brain
cells to form long fibrils, which then evolve into aggregates that
eventually destroy the cell. This has been linked to a several chemical
modifications of alpha-synuclein, one of which is nitration, where
nitrogen groups attach to its tyrosine amino acids.
However,
nitration of alpha-synuclein has proven hard to study in the lab. The
problem is that current nitrating methods generate non-homogeneous
mixtures of α-synucleins that are nitrated at different amino acid
sites. As a result, the proteins have different properties to natural
alpha-synuclein, and cannot be used effectively to mimic and study what
happens in Parkinson’s disease.
A new approach to dissecting nitration
Hilal
Lashuel’s team at EPFL has now developed the first technique to
produces homogeneous α-synucleins, where one or more of its tyrosines
can be properly nitrated. The scientists used a sophisticated protein
synthesis technique to make fragments of alpha-synuclein where nitrated
tyrosines could be incorporated and therefore nitrated independently
from each other. Because of this, it was also possible to control the
exact number or combinations of nitrated tyrosines, which offers a
powerful tool to study this process.
The fragments were then
recombined to produce the entire alpha-synuclein with correctly nitrated
tyrosines. Normally, this recombining involves a desulfurization step
that can ruin the attached nitrogen groups by adding electrons to them
(this is known as “reduction”). But lead author Ritwik Burai conceived
of a way to bypass the problem by modifying the desulfurization process.
The
new method produced a completely homogenously nitrated or tailored
mixture of nitrated alpha-synuclein. When tested in in vitro, the
modified proteins lost their ability to interact properly with vesicles,
such as those found in cells. Further tests also showed that
site-specific nitration changed alpha-synuclein’s structure, which
directly influences the protein’s tendency to form the aggregates seen
in Parkinson’s disease.
Tools that shape the future
Hilal
Lashuel’s team has been working on chemical modifications of
alpha-synuclein and other proteins for over five years. This method is
the latest achievement in this line, overcoming the last barrier,
nitration. “These advances allow us now to reconstruct, in vitro,
a-synuclein species with the same chemical properties as those isolated
from diseased human brains,” says Hilal Lashuel. Since the normal
functions of alpha-synuclein are still a mystery, being able to
reconstruct its chemical properties as they exist in the human brain may
also open new avenues for understanding its biological role, and how it
can become impaired in disease.
The new method is not limited to
alpha-synuclein, but can be used across different proteins and
different types of chemical modifications, which often underlie a
multitude of diseases. Just in the context of Parkinson’s disease,
Lashuel anticipates that the chemical tools his group has developed will
have an tremendous impact: “These advances will facilitate the
development of antibodies and imaging agents for the detection and
quantification of different a-synuclein species and aggregates along the
progression of Parkinson’s disease, which could lead to novel
approaches for its diagnosis and treatment.”
Reference
Burai R, Ait-Bouziad N, Chiki A, Lashuel HA. Elucidating
the role of site-specific nitration of α-synuclein in the pathogenesis
of Parkinson’s disease via protein semisynthesis and mutagenesis. JACS 13 March 2015. DOI: 10.1021/ja5131726