emin Xu

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.

Regulation of Brain G-protein Go by Alzheimer’s Disease Gene Presenilin-1

To investigate a possible association between G-proteins and presenilin-1 (PS-1), a series of glutathioneS-transferase-fusion proteins containing portions of PS-1 were prepared and used in vitro in binding experiments with tissue and recombinant G-proteins. The results demonstrate that the 39 C-terminal amino acids of PS-1 selectively bind the brain G-protein, Go. Addition of guanosine 5′-3-O-(thio)triphosphate promoted Godissociation from PS-1, indicating that this domain mimics the function of G-protein-coupling domains found in receptors. The 39-amino acid synthetic polypeptide activated Go in a magnesium ion-dependent manner. Physical interaction of full-length PS-1 and Go was also demonstrated. Following transfection of Goα and N-terminally FLAG-tagged PS-1 in COS-7 cells, Go was immunoprecipitated by FLAG antibodies. In addition, endogenous PS-1 and Goα were colocalized immunocytochemically in human glioma cell lines. The results indicate that PS-1 regulates Go activities in living cells.

Thrombin Rapidly Induces Protein Kinase D Phosphorylation, and Protein Kinase C δ Mediates the Activation

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKCδ phosphorylation and that the PKCδ-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKCδ mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKCδ inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKCε nor PKCζ affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKCδ in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKCδ in mediating thrombin-induced PKD activation in vascular smooth muscle cells.

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.

Identification of a Novel PSD-95/Dlg/ZO-1 (PDZ)-like Protein Interacting with the C Terminus of Presenilin-1

Presenilin-1 (PS-1) is the most causative Alzheimer gene product, and its function is not well understood. In an attempt to elucidate the function of PS-1, we screened a human brain cDNA library for PS-1-interacting proteins using the yeast two-hybrid system and isolated a novel protein containing a PSD-95/Dlg/ZO-1 (PDZ)-like domain. This novel PS-1-associated protein (PSAP) shares a significant similarity with a Caenorhabditis elegans protein of unknown function. Northern blot analysis revealed that PSAP is predominantly expressed in the brain. Deletion of the first four C-terminal amino acid residues of PS-1, which contain the PDZ domain-binding motif (Gln-Phe-Tyr-Ile), reduced the binding activity of PS-1 toward PSAP 4-fold. These data suggest that PS-1 may associate with a PDZ-like domain-containing protein in vivoand thus may participate in receptor or channel clustering and intracellular signaling events in the brain.

Lysophosphatidic Acid Induction of Tissue Factor Expression in Aortic Smooth Muscle Cells

Objective—Tissue factor (TF), the initiator of the coagulation cascade, is expressed by cells in atherosclerotic lesions. Lysophosphatidic acid (LPA) is a component of oxidized lipoproteins and an agent released by activated platelets. The present study investigated whether and how TF expression is regulated by LPA. Methods and Results—Northern blotting, Western blotting, and TF activity assays demonstrated that LPA markedly induced TF mRNA, protein, and activity in vascular smooth muscle cells. LPA-induced TF expression is primarily controlled at the transcriptional level. Phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signaling–regulated kinases (ERK1/2) was rapidly and markedly induced by LPA. MEK inhibitors U0126 and PD98059 blocked both ERK activation and the increase in TF mRNA. In contrast, the specific p38 MAP kinase inhibitor SB203580 had no effect on LPA-induced TF mRNA increase. The G&agr;i protein inhibitor, pertussis toxin, abolished LPA-induced phosphorylation of MEKs and ERKs, as well as the induction of TF mRNA. Conclusions—Our data demonstrate that a G&agr;i protein and activation of MEKs and ERKs mediate LPA-induced TF expression. Our data suggest that elevated LPA could be a thrombogenic risk factor by upregulating TF expression. These results may have important implications in vascular remodeling and vascular diseases.

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.

γ-Secretase Catalyzes Sequential Cleavages of the AβPP Transmembrane Domain

The biogenesis of the amyloid-beta peptide (Abeta) is a central issue in Alzheimers disease (AD) research. Abeta is produced by beta- and gamma-secretases from the amyloid-beta protein precursor (AbetaPP). These proteases are targets for the development of therapeutic compounds to downregulate Abeta production. gamma-secretase has received more attention 1) because it generates the C-terminus of Abeta, which is important in the pathogenesis of AD because the longer Abeta species are more amyloidogenic, and 2) because it cleaves AbetaPP within its transmembrane domain. In the understanding the mechanism of gamma-secretase cleavage, three major cleavage sites have been identified, namely, gamma-cleavage site at Abeta(40/42), zeta-cleavage site at Abeta(46), and epsilon-cleavage site at Abeta(49). Moreover, the novel finding that some of the known gamma-secretase inhibitors inhibit the formation of secreted Abeta(40) and Abeta(42), but cause an intracellular accumulation of long Abeta(46), provided information extremely important for the development of strategies aimed at the design of gamma-secretase inhibitors to prevent and treat AD. In addition, it has been established that the C-terminus of Abeta is generated by a series of sequential cleavages: first, epsilon-cleavage, followed by zeta-cleavage and finally by gamma-cleavage, commencing from the membrane boundary to the middle of the AbetaPP membrane domain.