Report on the IXth International Meeting on Cholinesterases
(Suzhou, China, May 6-10, 2007)
While the community of scientists working on cholinesterases is not formally established as a society, it regularly organizes international meetings approximately every 3 years. The IXth International Meeting on Cholinesterases was held in Suzhou (China) to acknowledge the strong and increasing contribution of Chinese scientists in the field of cholinesterase research. The meeting gathered more than 200 participants from 25 countries including 40 from China. ISN/IBRO provided grants for 21 overseas students.
The meeting was organized jointly by Prof. Karl W.K. Tsim (Hong Kong University of Science and Technology) and by Prof. Hua-Liang Jiang (Shanghai Institute of Materia Medica, Chinese Academy of Sciences) with the help of their colleagues and an International Advisory Committee. It was sponsored by Chinese universities, and companies, as well as foreign and international research Institutions (ISN, IBRO, IUBMB). The meeting was excellently organized in Suzhou, a city of about 4 million inhabitants located near Shanghai, which includes very modern parts with developing technological companies, and is also known for its very beautiful gardens, and for its ancient preserved water villages.
There were about 65 speakers and as in previous meetings, there were no parallel sessions so that the participants could attend all the lectures. This is important because the field of cholinesterase research is widely multidisciplinary, from very fundamental (molecular structure, catalytic mechanism, genetics and evolution, cell biology) to more applied aspects (natural and synthetic cholinesterase inhibitors in Alzheimer’s disease therapy, defense against anti-cholinesterase poisoning, pesticides and environment). Two novel features were included in the program: a roundtable on non classical functions and a structural session in which the 3D structure of cholinesterases could be viewed stereoscopically.
I would like to mention just a few of the many subjects that were discussed.
The crystallographic structure of Torpedo acetylcholinesterase was first determined by Sussman and his colleagues in 1991, and since then many structures of acetylcholinesterases from other species and of human butyrylcholinesterase have been obtained, with or without inhibitors occupying the active sites. In spite of differences all these structures are similar, and the occupation of the active site does not induce any major rearrangement. This is paradoxical, because this site can only be reached through a narrow “catalytic gorge” which seems to severely restrict the trafficking of substrates, of their hydrolytic products and of inhibitors, in contrast with the fact that the hydrolysis of acetylcholine by acetylcholinesterase is one of the fastest enzymatic reactions known and appears to be limited only by diffusion. It is therefore likely that the crystallographically determined structures represent constrained conformations and that the enzymes are in fact quite flexible in solution.
This question was approached by co-crystallization with analogs of substrates and reaction intermediates as well as kinetic-crystallography. It is remarkable that addition of an intracatenary disulfide bond may stabilize the enzyme without compromising its activity. It has long been shown that a fraction of newly synthesized acetylcholinesterase does not reach a catalytically active conformation, and it now seems possible to analyze this phenomenon, and in particular the role of chaperones such as protein disulfide isomerase.
The structure of the catalytic gorge has also been probed using enzymes from different species as templates in click chemistry. Bifunctional inhibitors binding both the peripheral site at the surface of the protein, and the buried active site have been obtained in this way: they possess extremely high affinity and reveal species differences. Strategies to obtain specific pesticides, directed for example against malaria-carrying mosquitoes and the detection of pesticides in food were discussed.
Bifunctional inhibitors with high affinity have also been synthesized because they might be useful therapeutic agents. Because the peripheral site may promote aggregation of the amyloid Ab peptide they might help to reduce its toxicity in Alzheimer’s disease. Some include moieties derived from huperzine A, a natural acetylcholinesterase inhibitor from a Lycoperdum used in Traditional Chinese Medicine. Such inhibitors may be beneficial for Alzheimer’s patients because they may increase the level of acetylcholine and therefore cholinergic activity in the brain. In addition, several participants showed that huperzine A and its derivatives may also act as neuroprotectants, particularly against the toxic effects of excitatory aminoacids, and the relationship between this effect and the anti-cholinesterase activity was discussed. Other compounds with mixed monoamine oxidase-cholinesterase inhibition and iron chelating properties seem particularly effective for neuroprotection (ladostigil).
The production and use of cholinesterases as protection against organophosphates (OPs) was extensively discussed. Cholinesterases act as bioscavengers, trapping the poison before it reaches its physiological target. Their protective effect would be greatly increased by converting them into OP-hydrolases which would then be able to catalytically inactivate a number of organophosphates. This has been attempted by directed mutagenesis, by introducing nucleophilic groups at appropriate positions, to allow their spontaneous reactivation after reaction with an OP. However, it is not certain that we sufficiently understand the mechanism of cholinesterases to predict the role of all residues, especially those of a second layer, which do not directly contact the substrates or inhibitors. Butyrylcholinesterase naturally hydrolyzes a wider range of substrates than acetylcholinesterase, including cocaine, and can be used to fight overdose and addiction.
The expression and regulation of cholinesterases, of specific variants, and of their anchoring proteins (collagen ColQ at neuromuscular junctions, the transmembrane protein PRiMA in the brain) have been largely discussed. Clearly, the function of cholinesterases requires a their strategic localization, which is achieved, in muscle and brain, by association of the “tailed” variant with ColQ or PRiMA.
The role of acetylcholinesterase in parasitic nematodes, in the resistance of insects to pesticides represent questions of both fundamental and applied interest. An analysis of cholinesterases in chordates offered an excellent illustration of gene evolution in the vertebrate lineage.
The roundtable discussion on alternative functions of cholinesterases was an important moment of the meeting. Several groups have reported that cholinesterases are involved in functions other than their classical role of neurotransmitter inactivation at cholinergic synapses. These roles are in fact quite diverse, ranging from the regulation of neurite extension, stress and autoimmune responses, apoptosis, to the etiology of Alzheimer’s disease through the promotion of amyloid peptide aggregation. The proposed mechanisms are equally diverse; some effects might be due to acetylcholine hydrolysis in non-synaptic contexts; others might be related to protein-protein interactions involving the catalytic domain, a possibility supported by the existence of homologous non-catalytic adhesion proteins such as neuroligin (neuroligin is involved in synaptogenesis, and mutations have recently been shown to cause autism). Yet other effects might be due to cleaved C-terminal peptides derived from acetylcholinesterase variants, expressed in normal brain (T) or induced by stress (R). In spite of a number of publications and review articles, the existence of non-classical functions, independent of the catalytic activity, is still debated.
It seems essential to base any discussion on accepted facts, i.e. results which have been independently reproduced in several laboratories. This is why the round table aimed at examining the relevant experimental data and if possible proposing critical approaches which might help to decide some controversial questions. There were some friendly but heated discussions about the reality of some effects. Clearly, the analysis of knock-out mice, in which either acetylcholinesterase, some of its variants, or butyrylcholinesterase have been entirely eliminated will be the key to answering many of these questions. It is surprising, and fortunate, that all these mutants can live, although sometimes with a severe phenotype.
In contrast with the rapid development of high throughput approaches, which yield presumably comprehensive lists of proteins involved in a given function, this meeting focused on just one type of protein. The importance and extreme diversity of the questions raised illustrates the great importance of in-depth studies on the catalytic mechanism of cholinesterases, and more generally on their regulation, conformation, and alternative variants and function(s). Cholinesterases offer a powerful tool to analyze cellular trafficking in the secretory pathway. In addition, cholinesterase research concerns fundamental and applied aspects such as population dynamics and pest control, as well as etiology and therapy of Alzheimer’s and other neurodegenerative diseases. The tenth meeting of the series will undoubtedly raise the same passionate interests and bring fresh questions and approaches. Like the first meeting, it will be held in Croatia.
more information about the meeting