Guozhang Mao

Identification of a New Presenilin-dependent ζ-Cleavage Site within the Transmembrane Domain of Amyloid Precursor Protein

γ-Secretase cleavage of β-amyloid precursor protein (APP) is crucial in the pathogenesis of Alzheimer disease, because it is the decisive step in the formation of the C terminus of β-amyloid protein (Aβ). To better understand the molecular events involved in γ-secretase cleavage of APP, in this study we report the identification of a new intracellular long Aβ species containing residues 1–46 (Aβ46), which led to the identification of a novel ζ-cleavage site between the known γ- and ϵ-cleavage sites within the transmembrane domain of APP. Our data clearly demonstrate that the new ζ-cleavage is a presenilin-dependent event. It is also noted that the new ζ-cleavage site at Aβ46 is the APP717 mutation site. Furthermore, we show that the new ζ-cleavage is inhibited by γ-secretase inhibitors known as transition state analogs but less affected by inhibitors known as non-transition state γ-secretase inhibitors. Thus, the identification of Aβ46 establishes a system to determine the specificity or the preference of the known γ-secretase inhibitors by examining their effects on the formation or turnover of Aβ46.

γ-Cleavage Is Dependent on ζ-Cleavage during the Proteolytic Processing of Amyloid Precursor Protein within Its Transmembrane Domain

β-Amyloid precursor protein apparently undergoes at least three major cleavages, γ-, ϵ-, and the newly identified ζ-cleavage, within its transmembrane domain to produce secreted β-amyloid protein (Aβ). However, the roles of ϵ- and ζ-cleavages in the formation of secreted Aβ and the relationship among these three cleavages, namely ϵ-, ζ-, and γ-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known γ-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Aβ46 is an intermediate precursor of secreted Aβ. Our co-immunoprecipitation data also reveal that, as an intermediate, Aβ46 is tightly associated with presenilin in intact cells. Furthermore, we identified a long Aβ species that is most likely the long sought after intermediate product, Aβ49, generated by ϵ-cleavage, and this Aβ49 is further processed by ζ- and γ-cleavages to generate Aβ46 and ultimately the secreted Aβ40/42. More interestingly, our data demonstrate that γ-cleavage not only occurs last but also depends on ζ-cleavage occurring prior to it, indicating that ζ-cleavage is crucial for the formation of secreted Aβ. Thus, we conclude that the C terminus of secreted Aβ is most likely generated by a series of sequential cleavages, namely first ϵ-cleavage which is then followed by ζ- and γ-cleavages, and that Aβ46 produced by ζ-cleavage is the precursor of secreted Aβ40/42.

The Novel Presenilin-1-associated Protein Is a Proapoptotic Mitochondrial Protein

Recent studies have suggested a possible role for presenilin proteins in apoptotic cell death observed in Alzheimers disease. The mechanism by which presenilin proteins regulate apoptotic cell death is not well understood. Using the yeast two-hybrid system, we previously isolated a novel protein, presenilin-associated protein (PSAP) that specifically interacts with the C terminus of presenilin 1 (PS1), but not presenilin 2 (PS2). Here we report that PSAP is a mitochondrial resident protein sharing homology with mitochondrial carrier protein. PSAP was detected in a mitochondria-enriched fraction, and PSAP immunofluorescence was present in a punctate pattern that colocalized with a mitochondrial marker. More interestingly, overexpression of PSAP caused apoptotic death. PSAP-induced apoptosis was documented using multiple independent approaches, including membrane blebbing, chromosome condensation and fragmentation, DNA laddering, cleavage of the death substrate poly(ADP-ribose) polymerase, and flow cytometry. PSAP-induced cell death was accompanied by cytochrome c release from mitochondria and caspase-3 activation. Moreover, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cell death, did not block the release of cytochrome c from mitochondria caused by overexpression of PSAP, indicating that PSAP-induced cytochrome c release was independent of caspase activity. The mitochondrial localization and proapoptotic activity of PSAP suggest that it is an important regulator of apoptosis.

Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein

Background: DR6-induced apoptosis mechanism is unknown. Results: DR6-induced apoptosis is dependent on cytochrome c release and Bax translocation, but is independent of caspase-8 and Bid. Conclusion: DR6-induced apoptosis is mediated by a unique pathway, different from type I and type II pathways. Significance: This study will lead to a better understanding of the mechanism by which DR6 induces apoptosis. Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) and the intrinsic pathway (the mitochondrial pathway). These two pathways can be linked by caspase-8-activated truncated Bid formation. Very recently, death receptor 6 (DR6) was shown to be involved in the neurodegeneration observed in Alzheimer disease. DR6, also known as TNFRSF21, is a relatively new member of the death receptor family, and it was found that DR6 induces apoptosis when it is overexpressed. However, how the death signal mediated by DR6 is transduced intracellularly is not known. To this end, we have examined the roles of caspases, apoptogenic mitochondrial factor cytochrome c, and the Bcl-2 family proteins in DR6-induced apoptosis. Our data demonstrated that Bax translocation is absolutely required for DR6-induced apoptosis. On the other hand, inhibition of caspase-8 and knockdown of Bid have no effect on DR6-induced apoptosis. Our results strongly suggest that DR6-induced apoptosis occurs through a new pathway that is different from the type I and type II pathways through interacting with Bax.

The same γ-secretase accounts for the multiple intramembrane cleavages of APP

It has been hypothesized that different C‐terminus of β‐amyloid peptide (Aβ) may be generated by different γ‐secretase activities. Recently, we have identified a new ζ‐cleavage site at Aβ46, leading to an important finding that the C‐terminus of Aβ is produced by a series of sequential cleavages. This finding prompted us to examine the effects of the known γ‐secretase inhibitors on different steps of the γ‐secretase‐mediated sequential cleavages and specifically their effects on the formation and turnover of the intermediate Aβ46. Our results demonstrate that some of the known inhibitors, such as L‐685,458 and III‐31C as well as inhibitors IV and V, inhibit the formation of secreted Aβ40/42 by inhibiting the formation of the intermediate Aβ46. However, most of the other inhibitors show no inhibitory effect on the formation of the intermediate Aβ46, but rather inhibit the turnover of Aβ46, resulting in its accumulation. In addition, the non‐steroidal anti‐inflammatory drugs (NSAIDs) ibuprofen and sulindac sulfide have no effect on the formation and turnover of Aβ46, but rather modulate the ratio of secreted Aβ at a step after the formation of Aβ40 and Aβ42. Thus, our data strongly suggest that the multi‐sequential intramembrane cleavages of amyloid precursor protein C (APP) are likely catalyzed by the same γ‐secretase.

Calpain inhibitor MDL28170 modulates Aβ formation by inhibiting the formation of intermediate Aβ46 and protecting Aβ from degradation

The observations that three major cleavages within the transmembrane domain of APP, namely, the γ‐cleavage, ε‐cleavage, and the newly identified ζ‐cleavage, are involved in the generation of secreted Aβ40 and Aβ42 prompted us to determine how the calpain inhibitor III MDL 28170 influences these three cleavages and Aβ formation. With the use of a cell culture system, our data demonstrate that 1) at either high concentrations, or at a low range of concentrations, at early time points, MDL 28170 inhibits the formation of secreted Aβ40 and Aβ42. However, this effect is due to inhibition of the intermediate Aβ46 generation by ζ‐cleavage and not due to direct inhibition of the γ‐cleavage that produces Aβ40/42 from Aβ46; 2) at low range of concentrations and at late time points, MDL 28170 causes an increase in secreted Aβ40/42 that likely results from inhibition of degradation of both the initial substrate, CTFβ, and the final product, Aβ40/42, of γsecretase. These data strongly suggest that formation of Aβ46 is a key step in the γ‐secretase mediated generation of Aβ40/42 and provide a new target for the development of Aβ inhibitors. These data also suggest that calpain and related proteases, which are sensitive to MDL 28170, play an important role in the accumulation of secreted Aβ.

Effects of γ-secretase cleavage-region mutations on APP processing and Aβ formation: interpretation with sequential cleavage and α-helical model

Overwhelming evidence supports the amyloid hypothesis of Alzheimer’s disease that stipulates that the relative level of the 42 amino acid β‐amyloid peptide (Aβ42) in relationship to Aβ40 is critical to the pathogenesis of the disease. While it is clear that the multi‐subunit gamma secretase is responsible for cleavage of the amyloid precursor protein (APP) into Aβ42 and Aβ40, the exact molecular mechanisms regulating the production of the various Aβ species remain elusive. To elucidate the underlying mechanisms, we replaced individual amino acid residues from positions 43 to 52 of Aβ with phenylalanine to examine the effects on the production of Aβ40 and Aβ42. All mutants, except for V50F, resulted in a decrease in total Aβ with a more prominent reduction in Aβ for residues 45, 48, and 51, following an every three residue repetition pattern. In addition, the mutations with the strongest reductions in total Aβ had the largest increases in the ratio of Aβ42/Aβ40. Curiously, the T43F, V44F, and T48F mutations caused a striking decrease in the accumulation of membrane bound Aβ46, albeit by a different mechanism. Our data suggest that initial cleavage of APP at the ε site is crucial in the generation of Aβ. The implicated sequential cleavage and an α‐helical model may lead to a better understanding of the γ‐secretase‐mediated APP processing and may also provide useful information for therapy and drug design aimed at altering Aβ production.

Pen-2 is dispensable for endoproteolysis of presenilin 1, and nicastrin-Aph subcomplex is important for both γ-secretase assembly and substrate recruitment

γ‐secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx‐defective 1 (Aph‐1), and presenilin enhancer 2 (Pen‐2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C‐terminal fragment (PS1C) caused by knockdown of pen‐2 or knockout of NCT or Aph‐1 was recovered by the addition of proteasome inhibitors, indicating that Pen‐2, as well as NCT and Aph‐1α, is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin‐Aph‐1 subcomplex plays not only an important role in γ‐secretase complex assembly but also in recruiting substrate C‐terminal fragment of amyloid precursor protein generated by β‐cleavage. Ablating any one component resulted in the instability of other components of the γ‐secretase complex, and the presence of all three of the other components is required for full maturation of NCT.

Both the N-terminal fragment and the protein-protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP.

Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.

The GxxxG motif in the transmembrane domain of AbetaPP plays an essential role in the interaction of CTF beta with the gamma-secretase complex and the formation of amyloid-beta

Gamma-secretase-mediated processing of the amyloid-beta protein precursor (AbetaPP) is a crucial step in the formation of the amyloid-beta peptide (Abeta), but little is known about how the substrate AbetaPP interacts with the gamma-secretase complex. To understand the molecular events involved in gamma-secretase-mediated AbetaPP processing and Abeta formation, in the present study we determined the role of a well conserved GxxxG motif in the transmembrane domain of AbetaPP. Our data clearly demonstrate that substitution of aspartic acid for the key glycine residues in the GxxxG motif almost completely abolished the formation of Abeta. Furthermore, our data revealed that substitution of aspartic acid for the glycine in this GxxxG motif disrupts the interaction of AbetaPP with the gamma-secretase complex. Thus, the present study revealed an essential role for the GxxxG motif in the interaction of AbetaPP with the gamma-secretase complex and the formation of Abeta.