CONFERENCE COVERAGE SERIES
Molecular Mechanisms of Neurodegeneration
University College, Dublin, Ireland
15 – 16 March 2005
Dublin: Warm Sun, Hot Science Fire up Meeting
March 14, the day after the AD/PD conference drew to a close in Sorrento, a meeting on Molecular Mechanisms of Neurodegeneration kicked off at University College Dublin, Ireland. Given that this island nation gets a bum rap for its supposedly bedraggling climate, it’s worth noting that those who hopped north from the lovely but cool Bay of Naples, and those who came direct, got to bask in bright sun and warm breezes. The science shone with top-notch presentations. This Irish scientist-turned-writer shared, with some satisfaction, a palpable sense among the participants that, at last, Ireland is a great place to do neuroscience.
Flush from a decade of unprecedented economic growth, Celtic coffers are now funding basic science at an appropriate level. Government is expected to back research and development to the tune of €2.5 billion ($3.4 billion) per year by 2006—not bad for a country with a population of fewer than four million people. (As a yardstick, the NIH budget in the U.S. was around $27 billion in 2003, in a country of 280 million). Part of the Irish funding goes to the relatively new Science Foundation Ireland. With some of its five-year grants approaching $5 million, its easy to see why Irish scientists are staying put and ex-pats like meeting organizer Dominic Walsh, as well as new faces such as Jochen Prehn, formerly of the Johann Wolfgang Goethe University, Frankfurt, Germany, have been lured to the Emerald Isle.
The story below is the first of a series of conference news we will post in the next few days.
Dublin: Chaperoned in Celtic Capital
It is great to have a chaperone when you visit a new city. At the meeting on Molecular Mechanisms of Neurodegeneration, held March 14-16 in Dublin, Ireland, chaperones were out in force.
Isabella Graef, Stanford University, opened the meeting by describing her efforts to bust plaques with small molecules that recruit larger chaperones. Preventing protein-protein interactions is a tall order for small molecules because they cannot hope to cover the vast number of contacts that can hold two proteins together. In the case of fibrils, the situation is exacerbated by the lack of “hotspots,” regions where the binding energy is concentrated. Breaking fibrils apart with a small molecule is a bit like asking a four-year-old to separate two boxers.
Graef’s solution is to have the pipsqueak bring along her heavyweight brother. Last October, Graef reported how a compound made by fusing the dye Congo red to a ligand for FK506 binding protein (FKBP) can prevent fibril formation. The compound, called SLF-CR for synthetic ligand for FKBP-Congo red, has enough affinity to bind to the fibrils and enough bulk, because it recruits FKBP, to interfere with fibril growth. In the presence of FKBP, the compound prevents fibril formation in cell-free systems and Aβ toxicity in cultured hippocampal neurons, though it has not yet been tested in vivo (see ARF related news story).
Graef hinted at how she might improve the plaque buster. Her strategy is to play with the three different modules of the compound: The targeting element, which binds the compound to Aβ fibrils; the recruiting element, which ropes in FKBP; and the linker that connects the two. In Dublin, Graef showed how other fibril-binding chemicals, such as the recently developed imidazole pyrimidine compound TZDM (see Kung et al., 2003), and the natural antioxidant curcumin, which may have plaque-busting talents of its own (see ARF related news story), may serve as better targeting elements. These two compounds cross the cell membrane, and bifunctional variants of TZDM, carrying either SLF or FK506, are even more cell-permeable, Graef reported. As for the recruiting element, she is currently working on substituting chaperones for FKBP, or cutting out the middle man altogether and recruiting a protease such as insulin-degrading enzyme or neprilysin, both of which degrade Aβ fibrils. Such a therapeutic strategy could prove beneficial not only for preventing plaque formation, but for clearing aggregates already formed, Graef suggested. As for the linker, Graef had already shown in the Science paper how lengthening it improved the potency several-fold to an IC50 of 50 nM.
The chaperone angle also marked the presentation of Linda Greensmith from University College London. Greensmith has been studying data on the use of small-molecule “co-inducers” of heat shock proteins. These co-inducers, such as arimoclomol, a hydroxylamine derivative, can amplify the heat shock response and so protect neurons against the toxic effects of mutant superoxide dismutase (SOD), which is responsible for about 20 percent of familial cases of the motor neuron disease amyotrophic lateral sclerosis (ALS).
Last spring, Greensmith’s group demonstrated that these co-inducers improve motor neuron survival and muscle strength in a mouse model of ALS and increase lifespan in these animals by about 25 percent (see Kieran et al., 2004). These effects are not just protective. Even when arimoclomol is given after the first symptoms have appeared, muscle strength improves and the mice survive about 18 percent longer than do littermates on placebo.
How, exactly, do these co-inducers work? In Dublin, Greensmith said that they may affect expression of key heat shock proteins. Her previous work had shown how BRX220, a derivative of the hydroxylamine bimoclomol, leads to increased expression of Hsp70 in astroglia when mice are subjected to acute injury to the sciatic nerve (Kalmar et al., 2002). The treatment also doubles the number of ventral horn neurons that survive injury. These neurons do more than merely hang on by the skin of their teeth, said Greensmith, as electrophysiological measurements showed that the number of working motor neuron contacts doubled in response to the drug.
Greensmith next posed the question of what causes the elevated Hsp70 expression? Could it relate to the finding that bimoclomol causes hyperphosphorylation and prolonged action of the major heat shock protein transcription factor, heat shock factor 1 (HSF-1) (Hargitai et al., 2003)? Possibly so, because when Greensmith examined mouse ventral horns from SOD mutant mice treated with arimoclomol, she found that HSF-1 was indeed hyperphosphorylated. What’s more, she found elevated expression of Hsp70 and Hsp90 in ventral horn neurons of these animals, but no changes in expression of Hsp27.
The pattern of heat shock protein expression in response to arimoclomol is interesting for two reasons. First, in an acute injury model, the effect on Hsp70 appeared in glia, not neurons. It is both plausible that the damage occurs faster than the ALS model, or that a totally different mechanism is at work, suggested Greensmith. Second, while Hsp70 and Hsp90 are known to be under the control of the HSF-1 promoter, Hsp27 is not. Therefore, the data fit nicely with a scenario whereby the hydroxylamine co-inducers act primarily on HSF-1, which in turn upregulates expression of Hsp70 and Hsp90. The question now is what causes increased phosphorylation of HSF-1. Meanwhile, it has become clear that the co-inducers are neuroprotective, said Greensmith.—Tom Fagan.
References
News Citations
- Busting up Plaques—Small Molecules Aided by Protein Heavies
- Curry Ingredient Spices Things Up by Blocking Aβ Aggregation
Paper Citations
- Kung MP, Skovronsky DM, Hou C, Zhuang ZP, Gur TL, Zhang B, Trojanowski JQ, Lee VM, Kung HF. Detection of amyloid plaques by radioligands for Abeta40 and Abeta42: potential imaging agents in Alzheimer's patients. J Mol Neurosci. 2003 Feb;20(1):15-24. PubMed.
- Kieran D, Kalmar B, Dick JR, Riddoch-Contreras J, Burnstock G, Greensmith L. Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice. Nat Med. 2004 Apr;10(4):402-5. PubMed.
- Kalmar B, Burnstock G, Vrbová G, Urbanics R, Csermely P, Greensmith L. Upregulation of heat shock proteins rescues motoneurones from axotomy-induced cell death in neonatal rats. Exp Neurol. 2002 Jul;176(1):87-97. PubMed.
- Hargitai J, Lewis H, Boros I, Rácz T, Fiser A, Kurucz I, Benjamin I, Vígh L, Pénzes Z, Csermely P, Latchman DS. Bimoclomol, a heat shock protein co-inducer, acts by the prolonged activation of heat shock factor-1. Biochem Biophys Res Commun. 2003 Aug 1;307(3):689-95. PubMed.
Further Reading
Papers
- de Cabo R, Fürer-Galbán S, Anson RM, Gilman C, Gorospe M, Lane MA. An in vitro model of caloric restriction. Exp Gerontol. 2003 Jun;38(6):631-9. PubMed.
Dublin: Targets, Therapies, and Trials
In terms of a treatment for Alzheimer disease, Aβ seems to be where it’s at these days. Some of the most promising strategies focus on preventing its production and oligomerization, or trying to enhance its clearance from the brain. At the Molecular Mechanisms of Neurodegeneration meeting in Dublin, all three possibilities were explored.
Grant Krafft, chairman and CSO of Acumen Pharmaceuticals, described his company’s strategy for tackling AD by targeting ADDLs, or Aβ-derived diffusible ligands. ADDLs—some would call them Aβ oligomers—are the real bad guys in AD, suggested Krafft, not misfolded proteins, fibrils, plaques, or even cell death. Misfolding is a misnomer, he contends, because at subnanomolar concentrations, thermodynamics favor the formation of Aβ assemblies. Though fibrils and plaques may not be good, they are not the root cause of AD either. And, while in late stages of the disease there is considerable loss of neurons, the real damage is done before cell death, and is caused by a breakdown in signal transduction, Krafft proposed. “How else could you have neurons packed with hyperphosphorylated tau?” he asked.
For all these reasons, Acumen, a relatively new kid on the biotech block, has focused on dealing with ADDL toxicity. Central to their strategy is data showing that ADDLs bind to synapses, particularly the synaptic cell surface receptor postsynaptic density 95 (PSD-95). This binding results in upregulation of intracellular signaling molecules including Arc and Rac (see Lacor et al., 2004): Arc is an immediate-early gene which has been linked to dysfunctional learning, while Rac activation has been linked to compromised long-term potentiation (LTP). Krafft reported that Acumen is using synaptosomes in high-throughput screens to find receptor antagonists that might prevent the toxic effect of these ADDLs.
Acumen is also developing molecules (in addition to antibodies) that interfere with ADDL assembly. Though interrupting protein-protein interactions presents quite a challenge to chemists (see ARF related Dublin story), in the case of Aβ42 it may be a little easier, suggested Krafft. The theory is that the two C-terminal amino acids, which are not present in the less “sticky” Aβ40, allow the formation of a β hairpin that then serves as the core of the ADDL structure. By targeting that hairpin it should be possible to develop drugs that prevent oligomerization of Aβ42. Acumen has obtained some lead compounds from high-throughput screens.
Krafft also described his collaboration with Bill Klein and chemist Chad Mirkin, both at Northwestern University, to develop antibodies that can be used to detect ADDLs in the CSF (see ARF related news story), and his ongoing cooperation with Merck & Co. to develop humanized antibodies that will selectively bind to ADDLs rather than Aβ monomers or fibrils.
Of course, given that Dublin serves as headquarters of Elan, the first company to bring an Alzheimer disease vaccine to clinical trial, it is probably fitting that at least some presentations would address the subject of AD and immunization. Elan’s vaccine program suffered a major setback when patients in a phase II clinical trial developed encephalitis. That disappointment has since been tempered by some encouraging results from those trials (see ARF immunotherapy update from the Sorrento AD/PD meeting). For example, postmortem examination of three patients who were injected with Elan’s AN-1792 has shown that the vaccine seems to dramatically reduce plaque burden. In Dublin, James Nicoll, a diagnostic pathologist at the University of Southampton General Hospital, England, showed that in a fourth patient there is evidence that plaques, though still present at death, were undergoing active removal (see Nicoll’s Sorrento presentation).
These postmortem results, plus tantalizing hints that the immunized patients do better cognitively and functionally (see ARF related news story), have spurred companies to develop second-generation passive and active immunotherapies that can circumvent the inflammatory response. Menelas Pangalos of Wyeth Pharmaceuticals, a partner in Elan’s vaccine program, reviewed some of the data coming out of Elan’s phase II trial (for a summary see coverage of Dale Schenk’s Sorrento presentation), but also hinted at ways to determine who can benefit from such therapy. Using transcriptional profiling, Pangalos and colleagues have been able to retrospectively “predict” which patients in the AN-1792 trial best respond to the vaccine. The profiling approach may also be useful for identifying unwanted side effects. Pangalos and colleagues have found that the transcriptional relationship between two specific genes is a good indicator of which patients in the Elan trial developed encephalitis.
Other therapeutic hopes are pinned on inhibiting β- and γ-secretases, the two proteases that sequentially cleave AβPP (see also related Sorrento news on γ-secretase and β-secretase). These strategies are not without controversy, however. One of the concerns about targeting γ-secretase is that not only does it cleave AβPP, but it also cleaves other transmembrane proteins including Notch, a major signaling molecule and a potential player in learning and memory (see ARF related news story and ARF news story). Some benzodiazepine derivatives, for example, inhibit γ-secretase but also cause goblet cell hyperplasia in the gut lining of the rat. This is thought to be caused by alteration of stem cell fate. The question on everyone’s mind, therefore, is whether a γ-secretase inhibitor can be developed that can prevent cleavage of AβPP but allow Notch cleavage to proceed as normal.
Mark Shearman reported that Merck Sharp & Dohme now have patents on three generic classes of γ-secretase inhibitor. These include compounds with IC50s in the low nanomolar range and which can reduce soluble Aβ in the brain of transgenic mice by 90 percent after a single dose. Though these inhibitors affect Notch and AβPP processing equally, he suggested that the “therapeutic window” between desirable and undesirable effects may be wide enough so that γ-secretase inhibitors can be used safely.
Shearman and colleagues have tested this in several ways, including molecular profiling. He reported how they used chip analysis of gene expression in the rat ileum to associate a specific gene expression profile to goblet cell hyperplasia. Then, when they tested benzodiazepine derivatives and their γ-secretase inhibitors on rat ileum, they found that the benzodiazepines give a huge hyperplasia profile, while one of their drug candidates, called compound F, showed very little signal.
In vivo data back up the profiling experiment. Shearman and colleagues have given the compound to transgenic mice daily for three months and seen no toxicity or Notch phenotype, even though the plaque load and area occupied by plaques were reduced by 60 and 40 percent, respectively. “So it is possible, with γ-secretase inhibitors, to obtain the efficacy that you want without the side effects based on Notch signaling,” said Shearman. He did caution, however, that rodents appear among the animals least sensitive to Notch-related effects.
Pangalos also reviewed the Wyeth γ-secretase program. The company has developed compounds that can inhibit the protease at IC50 values around 15 nM. In cell-based assays, these inhibitors prevent the release of Aβ42 and lead to an increase in levels of βCTFs produced by β-secretase.
In animals, a single dose (30mg/Kg) of one of the inhibitors, called GSI-1, causes a time-dependent inhibition of Aβ production, reducing synthesis by about 70 percent, reported Pangalos. Daily dosing at as little as 2.5mg/Kg can have a similar effect. As for side effects, even at the high doses there appeared to be little impact on thymocytes or the GI tract, he reported. As for behavior, when Pangalos and colleagues tested the compounds in transgenic mice (Tg2576), they found that the compounds can almost completely reverse defective fear responses.
Because γ-secretase shows a degree of promiscuity, β-secretases, or BACEs, have been considered by some to be more wholesome targets. There are two BACEs in the mammalian genome and we already know that one of them, BACE1, seems dispensable in mice (see ARF related news story). So what about BACE2?
Those bent on developing BACE inhibitors will be delighted to hear that BACE2 knockout mice are also viable, fertile, and seem no different from wild-type. In Dublin, Martin Citron, whose lab at Amgen Inc. discovered BACE1 in 1999 (see ARF related news story and Alzforum interview), reported his results with BACE2 knockouts and reported that BACE1/2 double knockouts are indistinguishable from wild-type animals, too.
The news may not come as a big surprise to many because BACE2 was always considered the minor β-secretase—its expression level is very low in neurons and overexpression of the protein actually leads to more α-secretase activity. The problem now will be to find a BACE inhibitor that can be turned into a useful drug. Citron reviewed the crystallographic data that shows the BACE1 active site as a very large binding pocket with as many as eight subsites. Some of the first-generation BACE inhibitors were bulky hexapeptides that could contact most of these subsites, but unfortunately these were not cell permeable, he said. Now, there are third-generation compounds available that are smaller, penetrate the cell, and can inhibit the protease while only binding to four of the subsites. “But as we still don’t know all the substrates for BACE, the question of toxicity is still one that needs careful attention,” he suggested.—Tom Fagan.
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References
News Citations
- Dublin: Warm Sun, Hot Science Fire up Meeting
- "Bio-Barcode" Amplifies Aβ Oligomer Signal, Proffers Candidate Diagnostic Test
- Sorrento: Immunotherapy Update Hot Off Lectern of AD/PD Conference
- Alzheimer’s Vaccine: In Some Patients, at Least, It Might Just Work
- Sorrento: Scientists Stop Clubbing, Start Tweaking, γ-Secretase
- Sorrento Secretase News: Baiting β, Awakening α
- The Senility-Presenilin Connection Turned Upside Down
- Notch and Memory: More Trouble for γ-Secretase Inhibition?
- More on BACE1, the New Darling of Alzheimer's Disease Drug Developers
- Presenting...β-Secretase
Paper Citations
- Lacor PN, Buniel MC, Chang L, Fernandez SJ, Gong Y, Viola KL, Lambert MP, Velasco PT, Bigio EH, Finch CE, Krafft GA, Klein WL. Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J Neurosci. 2004 Nov 10;24(45):10191-200. PubMed.
Other Citations
Further Reading
Dublin: The Tao of Tau
Followers of the Tao eastern philosophy advocate complete simplicity and naturalness; followers of the tau protein appreciate the latter, but can only wish for the former. At Molecular Mechanisms of Neurodegeneration, a focused meeting held in this western capital, there was a sense that tau might soon give up its secrets and enlighten us all. For though the protein is often enigmatic—is it good for microtubules or bad for microtubules?—we are closer than ever to attaining a fundamental understanding of tau, thanks in no small part to work from Eva-Maria Mandelkow's and Eckhard Mandelkow’s labs at the Max Planck Institute, Hamburg, Germany.
Of course, tau, a microtubule binding protein, is best known as the major component in the neurofibrillary tangles found in Alzheimer disease and other dementias. At the Dublin meeting, the Mandelkows (tau practitioners for many years) reviewed some recent revelations about the protein and strategies to prevent tangles.
It was apparent from Eva Maria’s presentation that to know tau is to understand time and space. In neurons, microtubules are essential for the transport of cargo between the cell body and the axons and dendrites. The Mandelkows previously demonstrated how tau, a large, natively unfolded protein that occupies a lot of hydrodynamic space, can inhibit this process—too much tau coats the microtubules, preventing access by other proteins such as kinesin, the motor that drives anterograde axonal transport (see ARF related news story). Their elegant video footage, familiar to meeting goers, showed that in N2A cells, elevated tau retards the anterograde movement of proteins, AβPP-carrying vesicles, and mitochondria away from the cell body and actually increases retrograde transport to the soma. The upshot is that neurites, starved for nourishment, shrivel and die.
Under normal conditions, cells are probably spared such ignominy by the controlled expression and phosphorylation of tau. Recently, the Mandelkows showed that microtubule affinity regulating kinase (MARK, also known as PAR1) can phosphorylate tau, making it yield its grip on microtubules (see ARF related news story). However, this process can go so far that the microtubules destabilize. For example, when CHO cells express MARK kinase (MARKK), MARK becomes highly active and the cells lose their microtubule network and rapidly apoptose, unless they are cultured in the presence of the microtubule stabilizer taxol, Eva-Maria reported. This pathway is at work endogenously in neuronal cells, she revealed, because when MARKK is knocked out in PC12 cells, they fail to differentiate when treated with nerve growth factor (NGF).
This on-again-off-again relationship between tau and microtubules does have a purpose. Eva-Maria showed how MARKK, MARK, and phosphorylated tau accumulate at the growth cone. Here there are practically no microtubules, but there is plenty of actin, and, indeed, when she stained growth cones for both phospho-tau and actin, she found that the two proteins co-localize. She proposed a scenario where phosphorylation of tau near the growth cone gives the microtubules enough temporary dynamism to advance toward the area of growth. Meanwhile, the phosphorylated tau, bound to the actin network in the cone, sits in wait for the incoming microtubule, which it then rejoins (presumably after being dephosphorylated).
How do these tau interactions relate to pathogenesis seen in various dementias? To answer this, Eva-Maria has made transgenic mice expressing human tau under control of the neural calmodulin kinase II (CaMK II) promoter and a tetracycline switch. The intention is to be able to control when and where tau is expressed and determine how age and tau expression may influence aggregation of the protein. Eva-Maria showed that when mice are reared with the promoter on, MARK phosphorylated tau (detected with the 12E8 antibody) is significantly increased in the pyramidal layer of the brain after three months. Tangle forming phospho-tau (PHF1 and AT8 antibodies) is not detectable, however, though the pathological forms of tau do appear later, at around 10 or 12 months, Mandelkow said. Because MARK phosphorylated tau is also the first to become elevated in Alzheimer brains, Mandelkow suggested that elevations in this form of the protein may be an omen that cells are trying to cope with a microtubule transport problem.
As for the influence of aging on tau aggregation, the animals that may answer that question are now only six-seven months old and Eva-Maria is waiting until they are older before she switches on human tau.
But what about the pathology? Can it be reversed? Perhaps, because Eva-Maria showed that in animals expressing human tau for nine months, a pan-tau antibody revealed dendrites packed with the protein, but when the promoter was then switched off for six weeks, the tau disappeared.
Eckhard Mandelkow also spoke to the possibility of reversing tau aggregation. First he addressed the question of why this intrinsically unstructured protein aggregates at all. Eckhard showed that tau shows very little secondary structure even when bound to microtubules, and it still has low intrinsic stability when it forms paired helical fragments (PHFs) —very low concentrations of guanidine hydrochloride, a commonly used protein denaturant, are sufficient to dissolve PHFs, he reported. This data, in fact, suggests that reversing tau aggregation might be easier than one would have thought.
So why does tau form PHFs? Last January, Eckhard showed that the aggregation of tau, like Aβ and many other fibrillogenic proteins, is driven by β-sheet formation (see von Bergen et al., 2005). In tau, the β-sheet originates from the repeat region, which is necessary for both microtubule binding and formation of paired helical fragments. Biophysical measurements, including infrared, fluorescence and circular dichroism spectroscopy, and x-ray diffraction, conducted at Eckhard’s lab indicate that two hexapeptide motifs in the second and third repeats can adopt a β-sheet structure, which also accompanies conversion of soluble to aggregated tau. Significantly, these two hexapeptides are close to the P301L and ΔK280 mutations that have been linked to familial cases of frontotemporal dementia. Currently, Eckhard is working in collaboration with Christian Griesinger at the Max-Planck-Institute, Gottingen, to study tau secondary structure using NMR. Spectra obtained from C13- and N15-labelled tau confirms that β structure is centered on the hexapeptide motifs in the repeats, Eckhard revealed. This work, still in progress, also confirms that the same amino acids are involved in both microtubule binding and PHF assembly, he reported.
Armed with the knowledge of how tau aggregates, Eckhard is now conducting high-throughput assays for chemicals that can inhibit or reverse tau aggregation. To date, over 200,000 compounds have been screened and of these, 1,266 inhibit PHF formation and 77 can break down pre-formed PHFs. These 77 were used in a homology search to find other potential PHF busters, and 241 of these are being studied in depth, he reported (for some examples, see Pickhardt et al., 2004). Eckard showed how these compounds reverse tau aggregation in N2a cells that are engineered with an inducible tau expression system. After about nine days, cells expressing a fast aggregating (ΔK280 mutation) tau have abundant PHFs and they also release lactate dehydrogenase (LDH), a sign of toxicity. The PHF busters stop aggregation and stem LDH release.
All told, data from the Mandelkows and others suggest that tau is all about yin and yang. Tau walks a fine line on microtubules. If that line is packed with tau, then axonal transport is blocked. In contrast, if there is not enough tau on the microtubules, then they fall apart. Excess or microtubule-free tau is also susceptible to aggregation, which can possibly be prevented in vivo with small molecules.—Tom Fagan.
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References
News Citations
- Tau Accused of Blocking Transport, Causing APP to Linger and Nerve Processes to Wither
- Tau Kinase Clears Microtubules—Keeps Axonal Transport on Track
Paper Citations
- von Bergen M, Barghorn S, Biernat J, Mandelkow EM, Mandelkow E. Tau aggregation is driven by a transition from random coil to beta sheet structure. Biochim Biophys Acta. 2005 Jan 3;1739(2-3):158-66. PubMed.
- Pickhardt M, Gazova Z, von Bergen M, Khlistunova I, Wang Y, Hascher A, Mandelkow EM, Biernat J, Mandelkow E. Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells. J Biol Chem. 2005 Feb 4;280(5):3628-35. PubMed.
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