Presenilin 1 stabilizes the C-terminal fragment of the amyloid precursor protein independently of gamma-secretase activity

The cleavage of the transmembrane amyloid precursor protein (APP) by beta-secretase leaves the C-terminal fragment of APP, C99, anchored in the plasma membrane. C99 is subsequently processed by gamma-secretase, an unusual aspartyl protease activity largely dependent on presenilin (PS), generating the amyloid beta-peptide (Abeta) that accumulates in the brain of patients with Alzheimer's disease. It has been suggested that PS proteins are the catalytic core of this proteolytic activity, but a number of other proteins mandatory for gamma-secretase cleavage have also been discovered. The exact role of PS in the gamma-secretase activity remains a matter of debate, because cells devoid of PS still produce some forms of Abeta. Here, we used insect cells expressing C99 to demonstrate that the expression of presenilin 1 (PS1), which binds C99, not only increases the production of Abeta by these cells but also increases the intracellular levels of C99 to the same extent. Using pulse-chase experiments, we established that this results from an increased half-life of C99 in cells expressing PS1. In Chinese hamster ovary cells producing C99 from full-length human APP, similar results were observed. Finally, we show that a functional inhibitor of gamma-secretase does not alter the ability of PS1 to increase the intracellular levels of C99. This finding suggests that the binding of PS1 to C99 does not necessarily lead to its immediate cleavage by gamma-secretase, which could be a spatio-temporally regulated or an induced event, and provides biochemical evidence for the existence of a substrate-docking site on PS1.     

Presenilin 1 modifies lipid raft composition of neuronal membranes

Protein-lipid interactions in the nervous system may provide insight into the causes of neurological disorders. In this study, we elucidated if expression of human presenilin 1 (PS1) in a mouse model changes the physico-chemical properties of brain membranes. PS1 is a multifunctional transmembrane protein and part of the γ-secretase complex. This complex is critical for the production of the Alzheimer related amyloid beta peptide. Brain membranes isolated from mice expressing a human wild-type PS1 transgene are less fluid and contain higher cholesterol and sphingomyelin levels. Moreover, our data reveal significant changes in membrane micro-domains and indicate that PS1 induces the formation of lipid rafts.     

Presenilin 2 expression in neuronal cells: induction during differentiation of embryonic carcinoma cells

Mutations in the presenilin 1 and 2 (PS1 and PS2) genes cause most cases of early onset Alzheimer's disease. The genes encode two homologous multipass membrane proteins. Since the endogenous expression of PS2 has been poorly analyzed to date, we studied PS2 expression and localization in cultured human neuroblastoma cells and mouse neuronal cells. PS2 was mainly detected as a full-length protein of about 52 kDa in these cells and in brain, in contrast to PS1 that is mainly detected as endoproteolytic N-terminal and C-terminal fragments. Using immunofluorescence we found that like PS1, PS2 colocalized with markers of the endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 and beta-COP. Double labeling for PS1 and PS2 indicated that both proteins are colocalized in neuroblastoma SH-SY5Y cells. To study PS2 expression during differentiation, mouse embryonic carcinoma P19 cells were treated with retinoic acid. We found minimal PS2 expression in undifferentiated cells, an increase from day 2, and a maximum at day 8 after treatment. PS1 expression remained constant during this period. The differential expression of PS1 and PS2 within the P19 cells following retinoic acid treatment indicates different utilization or temporal requirements for these proteins during neuronal differentiation.     

Selective reconstitution and recovery of functional gamma-secretase complex on budded baculovirus particles

In vitro reconstitution of functions of membrane proteins is often hampered by aggregation, misfolding, or lack of post-translational modifications of the proteins attributable to overexpression. To overcome this technical obstacle, we have developed a method to express multimeric integral membrane proteins in extracellular (budded) baculovirus particles that are released from Sf9 cells co-infected with multiple transmembrane proteins. We applied this method to the reconstitution of gamma-secretase, a membrane protease complex that catalyzes the intramembrane cleavage of beta-amyloid precursor protein to release Abeta peptides, the major component of amyloid deposits in Alzheimer brains as well as of Notch. When we co-infected Sf9 cells with human presenilin 1 (PS1), nicastrin, APH-1a, and PEN-2, a high-molecular-weight membrane protein complex that contained PS1 exclusively in its fragment form associated with three other cofactor proteins was reconstituted and recovered in a highly gamma-secretase-active state in budded virus particles, whereas nonfunctional PS1 holoproteins massively contaminated the parental Sf9 cell membranes. The relative gamma-secretase activity (per molar PS1 fragments) was concentrated by approximately 2.5 fold in budded virus particles compared with that in Sf9 membranes. The budded baculovirus system will facilitate structural and functional analyses of gamma-secretase, as well as screening of its binding molecules or inhibitors, and will also provide a versatile methodology for the characterization of a variety of membrane protein complexes.     

Presenilin 1 regulates pharmacologically distinct gamma -secretase activities. Implications for the role of presenilin in gamma -secretase cleavage

Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the gamma-secretase cleavage that generates the amyloid beta protein (Abeta). It is not clear how PSs regulate gamma-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the gamma-secretase cleavage, gamma-secretase themselves, or regulators of intracellular trafficking that indirectly influence gamma-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit Abeta production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct gamma-secretases, we show that PS1 regulates multiple pharmacologically distinct gamma-secretase activities as well as inducible alpha-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be gamma-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied.     

Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.     

Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles

Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.     

Characterization of presenilin-amyloid precursor interaction using bacterial expression and two-hybrid systems for human membrane proteins

An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of gamma-secretase are discussed.     

Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure.

Plant cell transformation by Agrobacterium tumefaciens involves the transfer of a single-stranded DNA-protein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VirB proteins were detected in the membrane fractions only. Three of these, VirB8, VirB9, and VirB10, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VirB proteins, VirB1, VirB4, and VirB11, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VirB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions.     

Presenilin-interacting proteins

Familial Alzheimer's disease (FAD) accounts for 5-10% of deaths from Alzheimer's disease (AD), and approximately 50% of these cases have been definitely linked to missense mutations in three genes, encoding the amyloid precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2). Of these, the vast majority of FAD-linked mutations are within PS1. There has been an extensive effort to identify proteins that functionally interact with PS1 and PS2 because of their clear roles in FAD. The goal of this review is to describe these proteins and to discuss in more detail the probable biological functions of a subset of the better-studied interacting proteins. In particular, the review examines APP, Notch, nicastrin, modifier of cellular adhesion (MOCA), beta-catenin, and the group of proteins involved in cell death, calcium metabolism and cell adhesion. We argue that, although a few of the interacting proteins are unambiguously involved in well-studied cellular pathways, their exact roles within these pathways have not been clearly defined, and indeed might vary between cell types. We also question the physiological relevance of some of the work linking PS to cell death pathways. Finally, we point out the value of using flies and worms to sort out the often contradictory work in the PS field, and we mention how knowledge of PS-interacting pathways will contribute to the development of new therapeutic strategies in AD.     

Presenilin/gamma-secretase and alpha-secretase-like peptidases cleave human MHC Class I proteins

HLA (human leucocyte antigen)-A2 is an MHC Class I protein with primary functions in T-cell development and initi-ation of immune cell responses. MHC I proteins also play roles in intercellular adhesion, apoptosis, cell proliferation and neuronal plasticity. By utilizing a sequence comparison analysis, we recently identified HLA-A2 as a potential substrate for the Alzheimer's disease-associated PS1 (presenilin 1)/gamma-secretase. alpha-Secretase-like membrane metalloproteinases are responsible for an initial shedding event, partially mediated by ADAM (a disinteg-rin and metalloproteinase)-10. Accordingly, activation or inhibition of alpha-secretase-like membrane metalloproteinases directly modulated levels of a 14 kDa HLA-A2 CTF (C-terminal frag-ment) in CHO (Chinese-hamster ovary) cells. To show that the HLA-A2 CTF is subsequently cleaved by PS1/gamma-secretase, we re-duced its activity in cell lines stably expressing HLA-A2 and in Jurkat T-cells expressing endogenous MHC I. Treatment with specific PS1/gamma-secretase inhibitors or expression of a dominant-negative construct led to a significant accumulation of HLA-A2 CTFs. We also identified the PS1/gamma-secretase cleavage product of HLA-A2 CTF, termed HLA-A2 intracellular domain, in cell-free and cell-based experiments. In the absence of proteasome inhibitors, HLA-A2 intracellular domain underwent rapid degrad-ation. These data indicate that MHC I proteins undergo extra-cellular domain cleavage mediated by alpha-secretases and the cleavage product is subsequently cleaved by PS1/gamma-secretase.     

Familial Alzheimer disease-linked presenilin 1 variants enhance production of both Abeta 1-40 and Abeta 1-42 peptides that are only partially sensitive to a potent aspartyl protease transition state inhibitor of "gamma-secretase"

Presenilin 1 (PS1) plays an essential role in intramembranous "gamma-secretase" processing of several type I membrane proteins, including the beta-amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted Abeta peptides and the effects of L-685,458, a potent gamma-secretase inhibitor, on inhibition of Abeta peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both Abeta1-42 and Abeta1-40 peptides. More surprisingly, whereas the IC(50) for inhibition of Abeta1-40 peptide production from cells expressing wild-type PS1 is approximately 1.5 microm, cells expressing the PS1deltaE9 mutant PS1 exhibit an IC(50) of approximately 4 microm. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of Abeta1-43 peptides are elevated in medium of PS1deltaE9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on gamma-secretase production of Abeta peptides and the disparity in sensitivity of these peptides to a potent gamma-secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of Abeta1-40 and Abeta1-42 peptides.     

Characterization of presenilin-amyloid precursor interaction using bacterial expression and two-hybrid systems for human membrane proteins

An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of γ-secretase are discussed.     

膜蛋白Presenilin 1的跨膜结构及催化机制

膜蛋白presenilin 1(PS1)是γ分泌酶的催化组分,是催化产生β淀粉样蛋白（β-amyloid,Aβ）的关键蛋白酶,因此也是治疗阿尔茨海默病（Alzheimer’s disease,AD）的主要靶点.PS1属于膜内裂解蛋白酶家族,这是一类在膜脂双层内部催化肽键水解断裂的蛋白酶.PS1其独特的跨膜结构和催化机制虽然还未完全揭示,但近期相关的研究取得了重要成果：PS1有10个疏水区,跨膜9次,其N端位于胞内,C端位于胞膜外或者内质网腔内,亦或不同程度地插入膜内,2个起催化作用的天冬氨酸残基都位于疏水性的膜内,膜蛋白底物被催化水解时必须先结合到酶的疏水表面上来,然后再进入位于活性部位.虽然PS1的晶体从未获得,但2006年首次解析的膜内裂解蛋白酶GlpG的晶体结构和所提出的催化机理为PS1催化机理的揭示奠定了基础,也为设计和筛选PS1/γ分泌酶的特异性抑制剂提供了理论依据.     

The membrane-bound proteins of periplasmic permeases form a complex. Identification of the histidine permease HisQMP complex

The membrane-bound proteins of periplasmic transport systems have been hypothesized to form a complex with relatively little experimental support. Here we present experimental evidence that HisQ, HisM, and HisP, the membrane-bound proteins of the periplasmic histidine transport system of Salmonella typhimurium, form such a complex. We have developed antibodies specific to each of these proteins to aid in their characterization. Extractions with urea, alkaline pH, or Triton X-114 show that HisQ and HisM are integral membrane proteins. By these tests HisP displays an unusual behavior, being associated with the membrane whether or not HisQ and HisM are present and despite its hydrophilic sequence. However, the nature of HisPs interaction with the membrane is shown to vary depending on the presence of HisQ and HisM. In their absence, HisP is somewhat peripherally associated with the membrane, while in their presence it binds much more tightly, indicating that it forms a complex in association with HisQ and HisM. This is demonstrated by the coimmunoprecipitation of all three proteins by antibodies directed against any one of them. Chemical cross-linking allowed the characterization of the subunit stoichiometry of the complex as two HisPs to one HisQ and one HisM. Within this complex all three proteins probably contact each other and the two HisPs form a dimer. We hypothesize that HisQ and HisM with their multiple membrane-spanning segments form a "channel" within which the HisP subunits are located.     

Differential effects of inhibitors on the gamma-secretase complex. Mechanistic implications

Gamma-secretase is a protease complex of four integral membrane proteins, with presenilin (PS) as the apparent catalytic component, and this enzyme processes the transmembrane domains of a variety of substrates, including the amyloid beta-protein precursor and the Notch receptor. Here we explore the mechanisms of structurally diverse gamma-secretase inhibitors by examining their ability to displace an active site-directed photoprobe from PS heterodimers. Most gamma-secretase inhibitors, including a potent inhibitor of the PS-like signal peptide peptidase, blocked the photoprobe from binding to PS1, indicating that these compounds either bind directly to the active site or alter it through an allosteric interaction. Conversely, some reported inhibitors failed to displace this interaction, demonstrating that these compounds do not interfere with the protease by affecting its active site. Differential effects of the inhibitors with respect to photoprobe displacement and in cell-based and cell-free assays suggest that these compounds are important mechanistic tools for deciphering the workings of this intramembrane-cleaving protease complex and its similarity to other polytopic aspartyl proteases.     

The nonconserved hydrophilic loop domain of presenilin (PS) is not required for PS endoproteolysis or enhanced abeta 42 production mediated by familial early onset Alzheimer's disease-linked PS variants

Presenilin 1 (PS1) and presenilin 2 (PS2) are polytopic membrane proteins that are mutated in the majority of early onset familial Alzheimer's disease (FAD) cases. Two lines of evidence establish a critical role for PS in the production of beta-amyloid peptides (Abeta). FAD-linked PS mutations elevate the levels of highly amyloidogenic Abeta ending at residue 42 (Abeta42), and cells with ablated PS1 alleles secrete low levels of Abeta. Several recent reports have shown that the hydrophilic loop (HL) domain, located between transmembrane domains 6 and 7, contains sites for phosphorylation, caspase cleavage, and sequences that bind several PS-interacting proteins. In the present report, we examined the metabolism of PS polypeptides lacking the HL domain and the influence of these molecules on Abeta production. We report that the deletion of the HL domain does not have a deleterious effect on the regulated endoproteolysis of PS, saturable accumulation of PS fragments, or the self-association of PS fragments. Abeta production was not significantly altered in cells expressing HL-deleted PS polypeptides compared with cells expressing full-length PS. Importantly, deletion of the HL domain did not affect FAD mutation-mediated elevation in the production of Abeta42. Furthermore, the deletion of the HL domain did not impair the role of PS1 or PS2 in facilitating Notch processing. Thus, our results argue against a biologically or pathologically relevant role for the HL domain phosphorylation and caspase cleavage and the association of PS HL domain-interacting proteins, in amyloid precursor protein metabolism and Abeta production or Notch cleavage.     

Studies of the role of ubiquitination in the interaction of ubiquilin with the loop and carboxyl terminal regions of presenilin-2

Ubiquilin was originally identified as a presenilin-interacting protein. We previously reported that ubiquilin interacts with both the loop and carboxyl terminus of presenilin proteins and that the ubiquitin-associated (UBA) domain of ubiquilin, which binds poly ubiquitin chains, is important for mediating this interaction. In the present study, we examined whether ubiquitination of presenilin-2 (PS2) is required for interaction with ubiquilin-1 by mutating lysine residues that may be targets for ubiquitination in the presenilin loop and carboxyl terminus regions. Mutation of two lysine residues in the PS2-loop region suggested that ubiquitination is not required for interaction with ubiquilin-1 and may, in fact, even negatively regulate the interaction. Similarly, we found that ubiquitination of the PS2 carboxyl terminus (PS2-C-terminus) is not required for interaction with ubiquilin-1, although our results suggest that it could play some role. Instead, we found that the mutation of either one of the two lysine residues in the carboxyl terminus of PS2 or the proline residues in the highly conserved PALP motif in this region results in destabilization of the mutant PS2 polypeptides because of increased degradation by the proteasome. Furthermore, by GST-pull-down assays we found that the mutant polypeptides were unable to bind ubiquilin, suggesting that loss of ubiquilin interaction leads to destabilization of presenilin polypeptides. Paradoxically, however, knockdown of ubiquilin expression by RNA interference did not alter the rate of turnover of PS2 proteins in cells. Instead, we found that PS2 synthesis was reduced, and PS2 fragment production was increased, suggesting that ubiquilin expression modulates biogenesis and endoproteolysis of presenilin proteins.     

Predicted membrane topology of the coronavirus protein E1

The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence.