Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active ␥-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the ␤-amyloid precursor protein (␤-APP) generating the amyloid ␤-peptide and the ␤-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. ␥-Secretase activity in isolated mitochondria was demonstrated using C83 (␣-secretasecleaved C-terminal 83-residue ␤-APP fragment from BD8 cells lacking presenilin and thus ␥-secretase activity) or recombinant C100-Flag (C-terminal 100-residue ␤-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the ␥-secretase inhibitors L-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of ␥-secretase activity to mitochondria.

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.

Background: Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

A cell-free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl^prolyl cis^trans isomerase PpiB was expressed in this system with dual amino acid-selective isotope labeling to identify the NMR signal from the active site-residue Arg87. Addition of the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide selectively shifted its 15 N-HSQC cross peak, con¢rming binding to the active site. As cell-free protein expression provides high yields of protein per unit mass of labeled amino acid and sample handling is minimal, this strategy presents an exceptionally inexpensive and rapid approach to protein analysis. ß

The ␥-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. ␥-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature ␥-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the ␥-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.

PRESENILIN1 (PSEN1) is the major locus for mutations causing familial Alzheimer's disease (FAD) and is also mutated in Pick disease of brain, familial acne inversa and dilated cardiomyopathy. It is a critical facilitator of Notch signalling and many other signalling pathways and protein cleavage events including production of the Amyloidβ (Aβ) peptide from the AMYLOID BETA A4 PRECURSOR PROTEIN (APP). We previously reported that interference with splicing of transcripts of the zebrafish orthologue of PSEN1 creates dominant negative effects on Notch signalling. Here, we extend this work to show that various truncations of human PSEN1 (or zebrafish Psen1) protein have starkly differential effects on Notch signalling and cleavage of zebrafish Appa (a paralogue of human APP). Different truncations can suppress or stimulate Notch signalling but not Appa cleavage and vice versa. The G183V mutation possibly causing Pick disease causes production of aberrant transcripts truncating the open reading frame after exon 5 sequence. We show that the truncated protein potentially translated from these transcripts avidly incorporates into very stable Psen1-dependent higher molecular weight complexes and suppresses cleavage of Appa but not Notch signalling. In contrast, the truncated protein potentially produced by the P242LfsX11 acne inversa mutation has no effect on Appa cleavage but, unexpectedly, enhances Notch signalling. Our results suggest novel hypotheses for the pathological mechanisms underlying these diseases and illustrate the importance of investigating the function of dominant mutations at physiologically relevant expression levels and in the normally heterozygous state in which they cause human disease rather than in isolation from healthy alleles.

Notch4 is a divergent member of the Notch family of receptors that is primarily expressed in the vasculature. Its expression implies an important role for Notch4 in the vasculature; however, mice homozygous for the Notch4(d1) knockout allele are viable. Since little is known about the role of Notch4 in the vasculature and how it functions, we further investigated Notch4 in mice and in cultured cells. We found that the Notch4(d1) allele is not null as it expresses a truncated transcript encoding most of the NOTCH4 extracellular domain. In cultured cells, NOTCH4 did not signal in response to ligand. Moreover, NOTCH4 inhibited signalling from the NOTCH1 receptor. This is the first report of cis-inhibition of signalling by another Notch receptor. The NOTCH4 extracellular domain also inhibits NOTCH1 signalling when expressed in cis, raising the possibility that reported Notch4 phenotypes may not be due to loss of NOTCH4 function. To better address the role of NOTCH4 in vivo, we generated a Notch4 null mouse in which the entire coding region was deleted. Notch4 null mice exhibited slightly delayed vessel growth in the retina, consistent with our novel finding that NOTCH4 protein is expressed in the newly formed vasculature. These findings indicate a role of NOTCH4 in fine-tuning the forming vascular plexus.

Tec is the prototypic member of a family of intracellular tyrosine kinases that includes Txk, Bmx, Itk, and Btk. Tec family kinases share similarities in domain structure with Src family kinases, but one of the features that differentiates them is a proline-rich region (PRR) preceding their Src homology (SH) 3 domain. Evidence that the PRR of Itk can bind in an intramolecular fashion to its SH3 domain and the lack of a regulatory tyrosine in the C terminus indicates that Tec kinases must be regulated by a different set of intramolecular interactions to the Src kinases. We have determined the solution structure of the Tec SH3 domain and have investigated interactions with its PRR, which contains two SH3-binding sites. We demonstrate that in vitro, the Tec PRR can bind in an intramolecular fashion to the SH3. However, the affinity is lower than that for dimerization via reciprocal PRR-SH3 association.