-Secretase Makes a Splash

Courtesy Helen Saibil Amyloid fibril structure obtained by cryo-electron microscopy with a cross-β structure modeled into the electron density map based on J.L. Jimenez et al., EMBO J, 18: 815–21, 1999 It started as a matter of guilt by association. Presenilin mutations have been blamed for most autosomal dominant forms of familial Alzheimer disease. And many had proposed that presenilin might play a role in the γ-secretase activity that cleaves amyloid precursor protein to create the plaque-forming amyloid β. When researchers finally pared down the exact actors involved in this proteolytic reaction, however, they found more than they bargained for. Since then, studies have refocused on the vagaries of this unique and seemingly important protease family. In this issue's Hot Papers, researchers from three separate groups demonstrated the minimal set of proteins required for γ-secretase activity. Nicrastrin (Nct), Aph-1, and Pen-2, when combined with a heterodimer of endoproteolysed presenilin fragments, form a stable, catalytically active, high-molecular-mass protein complex. This unusual aspartyl protease is the founding member of a novel class of proteases that carry out intramembrane cleavage, resulting in peptide-bond hydrolysis within the hydrophobic environs of the lipid bilayer.1 These novel proteases share similar catalytic residues with hydrophilic proteases, yet have devised a creative means of introducing water within the membrane. Moreover, γ-secretase's involvement in cleavage of Notch and type-I membrane receptors reveals an important role in signal transduction and developmental biology. "This protease is terribly important in biology – critical for signaling events in all multicellular organisms," explains biochemist Michael Wolfe of Harvard University and coauthor on one of the Hot Papers.3 "The Notch signaling pathway tells the cell what to be or not to be," says Wolfe. Thus, therapeutics aimed at inhibiting γ-secretase to stave off Alzheimer disease could have deleterious effects. Regardless, says Wolfe, "If we understand γ-secretase better, we can tweak it to stop making amyloid but not interfere with its normal function." PATHS OF MANY The first Hot Paper by Takeshi Iwatsubo and colleagues at the University of Tokyo identified the basic individual roles for the three additional proteins expressed in cells.2 Although presenilin's role as the catalytic center was suspected, the number and roles of other proteins thought to be involved were not. Evidence mounted implicating nicastrin, Aph-1 and Pen-2. "Everyone was reaching the same conclusion, and [the field] was progressing so quickly. So we were in a big hurry," recalls Iwatsubo, who says that one chance observation enabled them to reach their conclusions. "Our RNAi [interference] analysis showed that binding of Aph-1 and nicastrin to presenilin is an early event in gamma complex formation, providing the subcomplex with stability," he says. "Pen-2 finally activates γ-secretase by an unknown mechanism." They would not have made the discovery in time, he says, if not for the observation that Pen-2 depletion by RNAi resulted in accumulating immature presenilin. In contrast, depleting nicastrin or Aph-1 resulted in total loss of stabilized presenilin. "This finding gave us hints about the sequential complex-formation process of γ-secretase, and we could complete the work," he recalls. "[They] used a very clever strategy to determine the order of complex assembly and showed that Aph and Nct interacted with presenilin before Pen-2," says Raphael Kopan, a molecular biologist at Washington University in St. Louis. Together, these interactions prevented degradation of presenilin and facilitated its self-cleavage, he adds. Data derived from the Science Watch/Hot Papers database and the Web of Science (Thomson Scientific, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. "The role of presenilin cofactors in the γ-secretase complex," Takasugi N, Nature , 2003 Vol 422, 438-41 (Cited in 134 papers, Hist Cite Analysis)"γ-secretase is a membrane protein complex comprised of presenilin, nicastrin, aph-1, and pen-2," Kimberly WT, Proc Natl Acad Sci , 2003 Vol 100, 6382-7 (Cited in 106 papers, Hist Cite Analysis)"Reconstitution of γ-secretase activity," Edbauer D, Nat Cell Biol , 2003 Vol 5, 486-8 (Cited in 144 papers, Hist Cite Analysis) The second Hot Paper published by Dennis Selkoe, Michael Wolfe, and colleagues at Harvard Medical School and Brigham and Women's Hospital in Boston also showed the four proteins to be the minimal components required for γ-secretase activity.3 The three research groups had presented their data several months before publishing, says Selkoe. "We all knew each of the three labs was pursuing the concept." The competition nurtured a collegial and collaborative relationship between the three groups, "the way science should be done," he says. Selkoe and colleagues evaluated human homologs of Aph-1 and Pen-2 by tagging and expressing them in Chinese hamster ovary cells. Solubilized membrane preparations were precipitated using an affinity resin that specifically binds active presenilin/γ-secretase complexes, revealing that each associated with presenilin and nicastrin in an active complex. Subsequent coimmunoprecipitation experiments enabled them to pull down active γ-secretase by precipitation of either Aph-1 or Pen-2 individually. The precipitated beads were then used in an in vitro assay that confirms the presence of active γ-secretase activity by the generation of Aβ and tagged APP intra-cellular domain remnants, indicating cleavage. To confirm whether the four proteins were sufficient for activity, they then overexpressed them in combinations of three, comparing the effects to all four expressed together. As expected, only the four together demonstrated active γ-secretase activity. Subsequently, partial purification resulted in the four membrane proteins eluting together in a proteolytically active complex, confirmed by in vitro assay. The findings by Selkoe and colleagues, says Iwat-subo, "further clarified the early step of γ-secretase complex formation by showing that Aph-1 and nicastrin form the earliest subcomplex, which then binds presenilin." Finally, the third paper published by Harald Steiner, Christian Haass, and colleagues at Ludwig-Maximilians University in Munich sought to define the minimal set of components necessary for γ-secretase activity, by expressing human presenilin, nicastrin, Aph-1, and Pen-2 in the yeast, Saccharomyces cerevisiae.4 Although γ-secretase is readily demonstrated in mammalian cells, Steiner explains, one confounding problem is endogenous sources of proteins and cofactors. "But in yeast, there are no homologs of γ-secretase genes and no substrates," he says, adding that evidence of cleavage means the correct constitution of minimal components. To control for specificity, they coexpressed an inactive presenilin mutant with the same set of proteins. γ-secretase activity was monitored in yeast using a highly sensitive reporter-gene assay. As expected, wild-type presenilin that expressed with the other three proteins showed robust activity, whereas the mutant presenilin complex did not. Coexpression of triple protein combinations that lacked any one of the four components similarly failed to demonstrate activity. To confirm formation of a protein complex, the group then coimmunoprecipitated the proteins using antibodies specific to the active form of presenilin. The other three precipitated and the complex was shown to be active by in vitro assay. MEETING AT LAST Sangram Sisodia, molecular neurobiologist at the University of Chicago, says the findings published by Steiner, Haass, and colleagues are most compelling because yeast doesn't have homologs for these genes. "It's really a heterologous system," he says. Kopan agrees: "Haass and colleagues performed a critical experiment establishing that four proteins are required to reconstitute γ-secretase." But the ultimate proof, says Sisodia, will entail recreating the complex in vitro in an artificial lipid bilayer. IN THE BILAYER: © 2004 Bentham Science Publishers Ltd. Amyloid precursor protein (APP) is first cleaved by β-secretase to generate C99, the substrate for γ-secretase processing. C99 enters the active site of γ-secretase residing in presenilin, which together with Nct, Aph-1, and Pen-2 forms the γ-secretase complex. Two critical aspartates in transmembrane domains 6 and 7 catalyze the endoproteolytic cleavage which occurs at two topologically distinct sites (double arrowhead). Amyloid β liberated into extracellular space can form aggregates. (From H. Steiner, Curr Alz Res,1: 175–81, 2004.) Cell biologist Bart de Strooper of Catholic University Leuven in Belgium says the discovery has sparked intensive new research efforts. "These papers are, in fact, the end of the beginning of γ-secretase," he says. Sisodia says much work remains to be done. "We don't even know the stoichiometry of the complex, what it looks like, how active it is," he explains. "There is still no structural information about this complex. Who are the immediate neighbors? How do they interact and talk to each other, and what do they do?" Needless to say, an X-ray crystal structure of the complex is a long way off. Iwatsubo says his lab is currently focused on studying the structure-function relationship of γ-secretase. Notably, they're interested in figuring out what kind of abnormality in γ-secretase leads to increased cleavage at the 42nd position, resulting in the amyloid β42 species that more readily aggregate (cleavage at the 40th position is generally more common). Knowing this, he says, will pave the way for developing "selective" γ-secretase inhibitors for treating Alzheimer disease that won't interfere with Notch function. Wolfe says they are now studying γ-secretase from a structural angle. "We purified this protease and have done some electron microscopy and do have some low-resolution pictures of what this enzyme looks like," he says. Recent developments, says Steiner, indicate γ-secretase exhibits heterogeneous activity, with several complexes performing the same function. "We're not sure if individual complexes are processing multiple substrates," he says. Their research is now focused on addressing the functions of the individual subunits. "This solves the identity of a major mediator of signaling in all multicellular organisms and also as a major target for treating Alzheimer's disease," says Selkoe. The ultimate goal, says Wolfe, "is to try to pharmacologically modulate this protease so that it is not making amyloid anymore. If we understand it better, we can tweak it to stop making amyloid but not interfere with its normal function."

-Secretase Makes a Splash

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Nicole Johnston

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July 2005

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