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Weiming Xia

Publications and source records attributed to Weiming Xia.

14 recordsLinked to original sources
PubMed2004

Dimerization of Parkinson's disease-causing DJ-1 and formation of high molecular weight complexes in human brain.

Mutations in the DJ-1 gene have been implicated in the PARK7-linked autosomal recessive form of Parkinson's disease (PD). The molecular properties of DJ-1WT, DJ-1L166P, and a newly identified disease-causing mutant DJ-1M26I were explored after they were transiently expressed in mammalian cells. Treatment of intact, living cells with the chemical crosslinker disuccinimidyl suberate (DSS) revealed that DJ-1WT and mutant DJ-1M26I were present as stable homodimers; DJ-1L166P in particular tended to form high-order complexes as well. In contrast to DJ-1L166P that is quickly degraded by the proteasome, DJ-1M26I was found to be an efficiently expressed and stable variant of DJ-1, suggesting that these mutations have distinct biochemical effects on DJ-1. We further provide evidence that in human brain, under nondenaturing conditions, DJ-1 is present in high molecular weight (HMW) complexes of approximately 250-700 kDa containing parkin, another PD-associated protein.

Animals↗
PubMed2003

APP processing is regulated by cytoplasmic phosphorylation.

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.

Alzheimer Disease↗
PubMed2003

Intramembrane proteolysis by presenilin and presenilin-like proteases.

Regulated intramembrane proteolysis is a novel mechanism involving proteases that hydrolyze their substrates in a hydrophobic environment. Presenilin (PS) 1 and PS 2 are required for intramembrane cleavage of an increasing number of type I membrane proteins, including the amyloid precursor protein of Alzheimer's disease and the Notch receptor, which signals during differentiation and development. Mutagenesis, affinity labeling, biochemical isolation, and reconstitution in cells reveal that PS, in complex with co-factors nicastrin, APH-1 and PEN-2, apparently contains the active site of gamma-secretase, a novel membrane aspartyl protease. In addition, other related aspartyl proteases have been identified. These include members of the type-4 prepilin peptidase family in bacteria, which are known proteases and carry a GD motif conserved in PS. A group of multi-pass membrane proteins found in eukaryotes also contain YD and LGXGD motifs in two transmembrane domains that are conserved in PS and postulated to constitute an aspartyl protease active site. Among these is signal peptide peptidase (SPP), which cleaves remnant signal peptides derived from signal-peptidase-mediated ectodomain shedding. SPP cuts type II membrane proteins, illustrating that PS-like proteases play a key role in intramembrane proteolysis of single-pass membrane proteins oriented in either direction.

Alzheimer Disease↗
PubMed2003

Functional gamma-secretase complex assembly in Golgi/trans-Golgi network: interactions among presenilin, nicastrin, Aph1, Pen-2, and gamma-secretase substrates.

Gamma-secretase is a proteolytic complex whose substrates include Notch, beta-amyloid precursor protein (APP), and several other type I transmembrane proteins. Presenilin (PS) and nicastrin are known components of this high-molecular-weight complex, and recent genetic screens in invertebrates have identified two additional gene products, Aph1 and Pen-2, as key factors in gamma-secretase activity. Here, we examined the interaction of the components of the gamma-secretase complex in Chinese hamster ovary cells stably expressing human forms of APP, PS1, Aph1, and Pen-2. Subcellular fractionation of membrane vesicles and subsequent coimmunoprecipitation of individual gamma-secretase components revealed that interactions among all proteins occurred in the Golgi/trans-Golgi network (TGN) compartments. Furthermore, incubation of the Golgi/TGN-enriched vesicles resulted in de novo generation of amyloid beta-protein and APP intracellular domain. Immunofluorescent staining of the individual gamma-secretase components supported our biochemical evidence that the gamma-secretase components assemble into the proteolytically active gamma-secretase complex in the Golgi/TGN compartment.

Amyloid Precursor Protein Secretases↗
PubMed2003

Presenilin-1 exists in both pre- and post-Golgi compartments and recycles via COPI-coated membranes.

Presenilin-1 is involved in intramembrane proteolysis of various proteins, but its intracellular site of action has remained elusive. Here, we determined by quantitative immunogold-electron microscopy that presenilin-1 in Chinese hamster ovary cells is present in pre-Golgi compartments as well as at the plasma membrane and endosomes. Notably, a high percentage of presenilin-1 resides in COPI-coated membranes between the endoplasmic reticulum and the Golgi complex, indicating significant recycling to the endoplasmic reticulum. By contrast, the inactive aspartate mutant presenilin-1D257A is relatively excluded from COPI-coated membranes, concomitant with increased post-Golgi levels. These data provide critical evidence for the scenario that the complex containing presenilin-1 can serve as gamma-secretase at the plasma membrane or endosomes and suggest a role for COPI-mediated retrograde transport in regulating post-Golgi levels of presenilin-1.

Amino Acid Substitution↗
PubMed2003

Presenilin endoproteolysis mediated by an aspartyl protease activity pharmacologically distinct from gamma-secretase.

Presenilin (PS)-dependent gamma-secretase cleavage is the final proteolytic step in generating amyloid beta protein (A beta), a key peptide involved in the pathogenesis of Alzheimer's disease. PS undergoes endoproteolysis by an unidentified 'presenilinase' to generate the functional N-terminal and C-terminal fragment heterodimers (NTF/CTF) that may harbor the gamma-secretase active site. To better understand the relationship between presenilinase and gamma-secretase, we characterized the biochemical properties of presenilinase and compared them with those of gamma-secretase. Similar to gamma-secretase, presenilinase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC50 of approximately 1 microM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC50 < 1 microM) over gamma-secretase (IC50-16 microM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and gamma-secretase inhibition, suggesting that presenilinase, like gamma-secretase, is an aspartyl protease. Interestingly, several of the most potent gamma-secretase inhibitors (IC50 = 0.3 or 20 nM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of A beta in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permitted normal de novo generation of A beta and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from gamma-secretase.

Amyloid Precursor Protein Secretases↗
PubMed2003

Relationship between presenilinase and gamma-secretase.

Genetic and neuropathological studies suggest that processing of amyloid precursor protein (APP) to yield amyloid beta-protein (Abeta) plays an important role in the pathogenesis of Alzheimer's disease (AD). One of the current therapeutic efforts for AD is directed towards blocking the gamma-secretase activity that produces Abeta. Compelling evidence for presenilin (PS) possessing gamma-secretase activity includes a lack of Abeta production in PS knockout neurons and in cultured cells carrying a dominant negative mutation at either of two critical aspartate residues in PS, which may constitute the active site of gamma-secretase. In vitro studies have shown a binding of transition-state analog gamma-secretase inhibitors to PS N-terminal fragment (NTF) and C-terminal fragment (CTF), the functional form of PS detected in the high-molecular-weight gamma-secretase complex that also contains nicastrin, Aph-1 and PEN-2. Conversion of full-length PS into functional NTF and CTF is mediated by an unknown protease activity named presenilinase. Endoproteolysis of PS into NTF/CTF by presenilinase also requires two critical aspartate residues, suggesting that full-length PS may undergo autoproteolysis and PS itself is presenilinase. Similar to gamma-secretase, presenilinase seems to be an aspartyl protease, as aspartyl protease inhibitor pepstatin A is the most potent inhibitor toward presenilinase among different classes of protease inhibitors. While several well-characterized gamma-secretase inhibitors can block presenilinase activity in vivo and in vitro, the potency of inhibitors blocking presenilinase and gamma-secretase are not correlated. Lack of presenilinase inhibition by several potent gamma-secretase inhibitors suggests that these two protease activities are pharmacologically distinct.

Alzheimer Disease↗
PubMed2003

Amyloid inhibitors and Alzheimer's disease.

Neuritic plaques composed of amyloid beta-protein (A beta) are an early and invariant neuropathological feature of Alzheimer's disease (AD). The current preclinical search for drugs is mainly focused on decreasing A beta production by inhibiting beta- or gamma-secretase, blocking the formation of these plaques by preventing A beta protofibril and fibril formation, and alleviating the toxic effects of neuritic plaque deposition. Increasing numbers of drugs currently used as therapies for other diseases are now entering clinical trials for AD, but the molecular targets of these drugs and their relevance to A beta toxicity needs to be thoroughly addressed. This knowledge will allow us to fully understand the A beta-related pathways in AD pathogenesis and explore novel therapeutic interventions.

Alzheimer Disease↗
PubMed2002

Endoproteolysis of presenilin in vitro: inhibition by gamma-secretase inhibitors.

The final proteolytic step to generate the amyloid beta-protein (Abeta) of Alzheimer's disease (AD) from beta-amyloid precursor protein (APP) is achieved by presenilin (PS)-dependent gamma-secretase cleavage. AD-causing mutations in PS1 and PS2 result in a selective and significant increase in production of the more amyloidogenic Abeta42 peptide. PS1 and PS2 undergo endoproteolysis by an unknown enzyme termed presenilinase to generate the functional complex of N- and C-terminal fragments (NTF/CTF). To investigate the endoproteolytic activity that generates active PS, we used a mammalian cell-free system that allows de novo human PS NTF and CTF generation. PS NTF and CTF generation in vitro was observed in endoplasmic reticulum (ER)-enriched fractions of membrane vesicles and to a lesser extent in Golgi/trans-Golgi-network (TGN)-enriched fractions. AD-causing mutations in PS1 and PS2 did not alter de novo generation of PS fragments. Removal of peripheral membrane-associated and cytosolic proteins did not prevent de novo generation of fragments, indicating that presenilinase activity corresponds to an integral membrane protein. Among several general inhibitors of different protease classes that blocked the presenilinase activity, pepstatin A was the most potent inhibitor. Screening available transition state analogue gamma-secretase inhibitors led to the identification of two compounds that were able to prevent the de novo generation of PS fragments, with an expected inhibition of Abeta generation. Our studies provide a biochemical approach to characterize and identify this elusive presenilinase.

Amyloid Precursor Protein Secretases↗
PubMed2002

The search for gamma-secretase and development of inhibitors.

A considerable body of evidence has accumulated in recent years implicating the beta-amyloid protein (Abeta) in the etiology of Alzheimer s disease (AD). The highly hydrophobic Abeta can nucleate and form neurotoxic fibrils that are the principal components of the cerebral plaques characteristic of AD. Abeta is formed from the amyloid-beta precursor protein (APP) through two protease activities. First, beta-secretase cleaves APP at the Abeta N-terminus, resulting in a soluble, secreted APP derivative (beta-APPs) and a 12 kDa membrane-retained C-terminal fragment. The latter is further processed to Abeta by gamma secretases, which cleave within the single transmembrane region. Other APP molecules can be cleaved by alpha-secretase within the Abeta region, thus precluding Abeta formation. Both beta- and gamma- secretase have become prime targets for the development of therapeutic agent that reduce Abeta production. Beta-secretase has recently been identified as a new membrane-anchored aspartyl protease in the cathepsin D family. Inhibitor profiling, site-directed mutagenesis, and affinity labeling together have suggested that the multi-pass presenilins are gamma-secretases, novel intramembrane-cleaving aspartyl proteases activated through autoproteolysis. In this article, we review the current knowledge of gamma-secretase biochemistry and cell biology and the development of inhibitors of this important therapeutic target.

Alzheimer Disease↗
PubMedDate not supplied

Intracellular Abeta is increased by okadaic acid exposure in transfected neuronal and non-neuronal cell lines.

Intracellular Abeta was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Abeta immunostaining was enhanced, as demonstrated by two independent anti-Abeta antibodies. The confocal microscopic study revealed that the immunoreactivity of Abeta was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Abeta sandwich ELISA, showed significantly increased intracellular Abeta. False positive detection of Abeta by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Abeta, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Abeta.

Amyloid beta-Peptides↗
PubMedDate not supplied

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.

Amino Acid Sequence↗