24 March 2006. In yesterday’s Nature, Kun Ping Lu at Beth Israel Deaconess Medical Center, Boston, together with colleagues in the U.S., Taiwan, and China, reports that isomerization of a proline in the C-terminal end of amyloid-β precursor protein (AβPP) reduces production of amyloid-β (Aβ). The conformational change is catalyzed by the prolyl isomerase Pin1, which may also prevent neurofibrillary tangles by isomerizing a proline residue in the microtubule binding protein tau (see ARF related news story). The actions of Pin1 on both AβPP and tau suggest that the prolyl isomerase might play a central role in AD pathology.
Proline, the sole α-imino acid in nature’s protein repertoire, is the only peptide residue that can flip-flop between two isomeric conformations, cis and trans. Such isomerizations have a profound effect on the shape of the protein backbone and in the late 1990s were predicted to be the basis for Pin1’s essential role in mitosis. Pin1 has since been linked to isomerization of a number of different proteins, including tau; transcription factors c-jun, NF-κB, and β-catenin; and the cell cycle protein cyclin D1. It has also been shown to bind to a plethora of other proteins, including, most recently, the neuronal harbinger of death BIM(EL), which triggers apoptosis. Whether Pin1 actually catalyzes proline isomerization in these binding partners remains to be determined.
In fact, proving that Pin1 causes proline isomerization has been a technical nightmare. Though miniscule amounts of the enzyme can elicit conformational changes in proteins such as Cdc25 (see Stukenberg and Kirschner, 2001), no one has conclusively demonstrated that these are caused by a cis/trans isomerization. But in collaboration with NMR spectroscopist Linda Nicholson at Cornell University, Lu and colleagues have now been able to visualize that flip-flop in full-length AβPP. Using ROESY (rotational frame Overhauser effect spectroscopy) NMR, they demonstrate the presence of both cis and trans isomers of proline 669, and transitions between the two isomers catalyzed by small amounts of Pin1.
Test tube experiments are one thing, but does this isomerization have physiological significance? Last year, Japanese researchers led by Takafumi Uchida at Tohoku University, showed that Pin1 binds to a threonine 668-proline 669 fragment in C99, the truncated form of AβPP that results from β-secretase cleavage of the full-length protein (see Akiyama et al., 2005). This was no surprise given that phosphorylated serine/threonine-proline is a well-known binding motif for the isomerase. Now, joint first authors Lucia Pastorino, Anyang Sun, Pei-Jung Lu, and their colleagues report that the isomerase also binds to full-length AβPP. They also tested how Pin1 affects processing of the full-length precursor protein in cell culture and in mice expressing the human AβPP carrying the Swedish double mutation (Tg2576).
The authors found that in Chinese hamster ovary (CHO) and H4 neuroglioma cells, AβPP and Pin1 colocalize and coimmunoprecipitate. They also found that overexpression of Pin1 reduces the amount of Aβ secreted from the CHO cells. In contrast, when the authors knocked out Pin1 in a breast cancer cell line, the cells produced about threefold less sAPPα, the soluble N-terminal fragment released by α-secretase. Taken together, the data seems to suggest that Pin1 reduces formation of Aβ by increasing the non-amyloidogenic processing of AβPP mediated by α-secretase.
In contrast, Uchida’s group had found that Pin1 increased production of Aβ from C99 when both were expressed in murine embryonic fibroblasts. Because C99 is poorly cleaved by α-secretase, this experiment might miss any effect Pin1 exerts on α-secretase-mediated AβPP processing. Nevertheless, the different effects seen on full-length versus β-secretase cleaved precursor suggest that the role of Pin1 in AβPP processing may be complex.
Pastorino and colleagues used Pin1-/- mice generated by Uchida’s lab to examine the relationship between the isomerase and AβPP processing in vivo. The Japanese group had found that soluble and insoluble Aβ40/42 were lower in Pin1-negative mice than in wild-type, though they did not report at what age they tested the animals. Now, Pastorino and colleagues report that while young (i.e., 2-6 months old), Pin1-negative mice had about the same amount of Aβ in the brain as wild-type littermates; in older animals (15 months), the absence of the isomerase caused about a 30 percent increase in the amount of insoluble Aβ42 (levels of soluble Aβ and insoluble Aβ40 stayed normal). It should be noted that this increase is relatively mild compared to some mouse models of AD. Tg2576 animals, for example, produce over 10-fold more Aβ42 than do wild-type mice and have abundant Aβ plaques. Tg2576 and other animal models also have well-documented learning and memory problems, so it will be interesting to see how the Pin1-/- animals fare in behavioral studies, too.
To test if the mild increase in insoluble Aβ in the Pin1-negative mice is due to some nonspecific, age-related phenomenon, the authors examined what happens to Tg2576 mice when Pin1 is absent. They found that by 6 months, Tg2576/Pin1-/- mice produced about 50 percent more insoluble Aβ42 than did age-matched transgenic littermates, suggesting that Pin1 may have a direct effect on Aβ production in vivo.
How could isomerization of proline 669 in the C-terminal, intracellular end of AβPP affect processing by α- and/or β-secretase on the extracellular side of the membrane or even by γ-secretase in the transmembrane domain? “That’s a very interesting question. We don’t know for sure, but we think it may be related to proteins that control trafficking of AβPP by binding to its C-terminal end,” said Lu.
Fe65, for example, which suppresses Aβ production, cannot bind to AβPP if threonine 668 is phosphorylated (see Ando et al., 2001), but dephosphorylation at that position requires that proline 669 be in the trans form. The natural, albeit slow, isomerization of the proline residue would forever protect a small portion of the total AβPP from dephosphorylation at threonine 668, increasing the chances for β- and subsequent γ-cleavage. “We believe that the role of Pin1 is to reset any cis-proline 669 to trans, so that the threonine can be dephosphorylated,” suggested Lu
This theory fits in with other observations. Li-Huei Tsai’s group at Harvard University has shown that phosphorylation of threonine 668 leads to increased production of Aβ in cell lines and that more of the phosphorylated form of the amino acid is found in tissue samples from AD brain (see Lee et al., 2003). Perhaps the big question is: How does threonine 668 get phosphorylated to begin with? It turns out that more of the amino acid is bound to phosphate in mitotic cells. This is curious, given Pin1’s role in mitosis and in light of a hypothesis suggesting that foiled attempts at cell cycle re-entry may be a trigger for neurodegeneration (see related ARF related Live Discussion). Stress may also lead to phosphorylation of threonine 668, suggested Lu.
The actions of Pin1 are likely to be multifaceted. It seems to function in the proliferation of breast cancer cells, and apoptosis mediated by mitochondria (see Becker and Bonni, 2006), yet without it, neurons are prone to neurodegeneration. As with most research, knowledge of Pin1 will benefit from distribution of the Pin1-/- mice and further exploration by other labs in the field.—Tom Fagan.
Pastorino L, Sun A, Lu P-J, Zhou XZ, Balastik M, Finn G, Wulf G, Lim J, Li S-H, Li X, Xia W, Nicholson LK, Lu KP. The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-β production. Nature. March 23, 2006;440:528-534. Abstract