Abstract
Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.
Highlights
Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for treating Parkinson disease
In answering the second question above, we demonstrated that recombinant CDNF protects and rescues both primary cultures of dopaminergic neurons, which were isolated from E14 mouse mesencephalon, and cultures of differentiated neuron 2a (N2a) cells from toxicity induced by ␣-synuclein oligomers
To obtain the full-length protein, a new protocol for recombinant expression and purification of FL-CDNF was developed by our group, which included an additional step of Zn2ϩ affinity chromatography to eliminate any metalloprotease activity that might explain why CDNF undergoes a spontaneous cleavage near position 105, freeing the C terminus
Summary
Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for treating Parkinson disease. The three-dimensional crystal structure of the N terminus (residues 9 –107) of CDNF has been determined, but there have been no published structural studies on the fulllength protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. The synergic effect of MANF and CDNF intranigral overexpression in a rat model for PD supports this hypothesis [21] It is still not clear why the crystallized structure of CDNF lacks a 6-kDa C-terminal domain, which was intriguingly cleaved either during or after purification even when the solution of the protein appeared to be pure [14, 22]. In answering the second question above, we demonstrated that recombinant CDNF protects and rescues both primary cultures of dopaminergic neurons, which were isolated from E14 mouse mesencephalon, and cultures of differentiated neuron 2a (N2a) cells from toxicity induced by ␣-synuclein oligomers
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