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.     

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.     

Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age

Oxidative stress is strongly implicated in the progressive decline of cognition associated with aging and neurodegenerative disorders. In the brain, free radical-mediated oxidative stress plays a critical role in the age-related decline of cellular function as a result of the oxidation of proteins, lipids, and nucleic acids. A number of studies indicate that an increase in protein oxidation and lipid peroxidation is associated with age-related neurodegenerative diseases and cellular dysfunction observed in aging brains. Oxidative stress is one of the important factors contributing to Alzheimer's disease (AD), one of whose major hallmarks includes brain depositions of amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP). Mutation in APP and PS-1 genes, which increases production of the highly amyloidogenic amyloid beta-peptide (Abeta42), is the major cause of familial AD. In the present study, protein oxidation and lipid peroxidation in the brain from knock-in mice expressing human mutant APP and PS-1 were compared with brain from wild type, as a function of age. The results suggest that there is an increased oxidative stress in the brain of wild-type mice as a function of age. In APP/PS-1 mouse brain, there is a basal increase (at 1 month) in oxidative stress compared to the wild type (1 month), as measured by protein oxidation and lipid peroxidation. In addition, age-related elevation of oxidative damage was observed in APP/PS-1 mice brain compared to that of wild-type mice brain. These results are discussed with reference to the importance of Abeta42-associated oxidative stress in the pathogenesis of AD.     

Two isoforms of PSAP/MTCH1 share two proapoptotic domains and multiple internal signals for import into the mitochondrial outer membrane

Presenilin 1-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed. presenilin 1-associated protein-mitochondrial carrier homolog 1; mitochondria; apoptosis; presenilin; Alzheimer's disease     

ProSAP-interacting protein 1 (ProSAPiP1), a novel protein of the postsynaptic density that links the spine-associated Rap-Gap (SPAR) to the scaffolding protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the "Fezzins," which adds further complexity to the organization of the PSD protein network.     

nRap GEP: a novel neural GDP/GTP exchange protein for rap1 small G protein that interacts with synaptic scaffolding molecule (S-SCAM).

Synaptic scaffolding molecule (S-SCAM) has six PDZ domains through which it interacts with N-methyl-d-aspartate receptors and neuroligin at synaptic junctions. We isolated here a novel S-SCAM-binding protein. This protein has one PDZ, one Ras association, one Ras GDP/GTP exchange protein (Ras GEP) domain, and one C-terminal consensus motif for binding to PDZ domains. We named it nRap GEP (neural Rap GEP). nRap GEP moreover has an incomplete cyclic AMP (cAMP)-binding (CAB) domain. The domain organization of nRap GEP is similar to that of Epac/cAMP-guanine nucleotide exchange factor (GEF) I, except that Epac/cAMP-GEFI has complete CAB and Ras GEP domains but lacks the other two domains and the C-terminal motif. nRap GEP showed GEP activity for Rap1 but did not bind cAMP. nRap GEP was specifically expressed in rat brain. Immunohistochemical analysis revealed that nRap GEP and S-SCAM were localized at synaptic areas of the cerebellum. These results suggest that nRap GEP is a novel neural Rap1-specific GEP which is associated with S-SCAM.     

Oxidative Stress in Synaptosomal Proteins from Mutant Presenilin-1 Knock-in Mice: Implications for Familial Alzheimer's Disease

Presenilin-1 (PS-1) is a transmembrane protein that may be involved in the processing of amyloid precursor protein (APP). Mutations in PS-1 are the major cause of familial Alzheimer's disease (AD). AD brain is under significant oxidative stress, including protein oxidation. In the present study, protein oxidation was compared in synaptosomes from knock-in mice expressing mutant human PS-1 (M146V mutation) and from wild-type mice expressing non-mutant human PS-1. Synaptosomal membrane protein conformational alterations associated with oxidative stress were measured using electron paramagnetic resonance (EPR) in conjunction with a protein-specific spin-label. Direct synaptosomal protein oxidation was assessed by a carbonyl detection assay. Synaptosomal proteins from PS-1 mutant mice displayed increased oxidative stress as measured by both techniques, compared with synaptosomal proteins from wild type mice. These data suggest that PS-1 mutations cause oxidative alterations in synaptosomal membrane protein structure and oxidative modification of synaptosomal proteins. Our findings suggest that familial AD may be associated with oxidative stress that may play a pivotal role in neuronal dysfunction and death.     

Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.     

PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/delta-catenin and p0071

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.     

Exposure of any of two proapoptotic domains of presenilin 1-associated protein/mitochondrial carrier homolog 1 on the surface of mitochondria is sufficient for induction of apoptosis in a Bax/Bak-independent manner

Presenilin 1-associated protein/mitochondrial carrier homolog 1 (PSAP/Mtch1) is a proapoptotic outer mitochondrial membrane protein first identified as a presenilin 1-associated protein. The mechanism by which it induces apoptosis upon overexpression in cultured cells is so far unknown. We had previously reported that deletion of two independent regions of PSAP/Mtch1 is required to prevent apoptosis. We now report that mitochondrial targeting of the region containing both proapoptotic domains, or any of them independently, to the outer membrane is sufficient to induce apoptosis. On the other hand, targeting of that region to the surface of the endoplasmic reticulum does not induce apoptosis, indicating that attachment of those domains to the outer mitochondrial membrane, and not just cytosolic exposure, is a requisite for apoptosis. Overexpression of PSAP/Mtch1 in cultured cells causes mitochondrial depolarization and apoptosis that does not depend on Bax or Bak, since apoptosis is induced in mouse embryonic fibroblasts lacking these two proteins. Our results suggest that apoptosis induced by PSAP/Mtch1 likely involves the permeability transition pore.     

The ERBB2/HER2 receptor differentially interacts with ERBIN and PICK1 PSD-95/DLG/ZO-1 domain proteins

Identification of protein complexes associated with the ERBB2/HER2 receptor may help unravel the mechanisms of its activation and regulation in normal and pathological situations. Interactions between ERBB2/HER2 and Src homology 2 or phosphotyrosine binding domain signaling proteins have been extensively studied. We have identified ERBIN and PICK1 as new binding partners for ERBB2/HER2 that associate with its carboxyl-terminal sequence through a PDZ (PSD-95/DLG/ZO-1) domain. This peptide sequence acts as a dominant retention or targeting basolateral signal for receptors in epithelial cells. ERBIN belongs to the newly described LAP (LRR and PDZ) protein family, whose function is crucial in non vertebrates for epithelial homeostasis. Whereas ERBIN appears to locate ERBB2/HER2 to the basolateral epithelium, PICK1 is thought to be involved in the clustering of receptors. We show here that ERBIN and PICK1 bind to ERBB2/HER2 with different mechanisms, and we propose that these interactions are regulated in cells. Since ERBIN and PICK1 tend to oligomerize, further complexity of protein networks may participate in ERBB2/HER2 functions and specificity.     

The calcium-independent receptor for alpha-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins

Subtypes of the calcium-independent receptors for alpha-latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1. In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.     

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 Alzheimer's 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.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Identification of proteins interacting with the rat somatostatin receptor subtype 2.

Using the yeast two hybrid system we have identified a novel protein termed somatostatin receptor interacting protein (SSTRIP) from human brain which interacts with the rat somatostatin receptor subtype 2. Interaction with the receptor C-terminus is mediated by a PSD-95/discs large/ZO-1 (PDZ) domain which exhibits high similarity to the PDZ domain of cortactin binding protein 1 (CortBP1). SSTRIP and CortBP1 define a novel family of multidomain proteins containing ankyrin repeats, SH3- and SH3 binding regions and a sterile alpha motif (SAM domain) in addition to the PDZ domain. Both SSTRIP and CortBP1 can be co-immunoprecipitated with the somatostatin receptor when co-expressed in HEK cells. Interestingly, co-localization of SSTR2 and CortBP1 at the plasma membrane is increased when SSTR2 is stimulated by agonists.     

Phosphorylation of PDZ1 domain attenuates NHERF-1 binding to cellular targets

NHERF-1 (Na(+)-H(+) exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including beta(2)-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.     

The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump

The Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here, we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons.     

Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking

Palmitoylation, a key regulatory mechanism controlling protein targeting, is catalyzed by DHHC-family palmitoyl acyltransferases (PATs). Impaired PAT activity is linked to neurodevelopmental and neuropsychiatric disorders, suggesting critical roles for palmitoylation in neuronal function. However, few substrates for specific PATs are known, and functional consequences of palmitoylation events are frequently uncharacterized. Here, we identify the closely related PATs DHHC5 and DHHC8 as specific regulators of the PDZ domain protein GRIP1b. Binding, palmitoylation, and dendritic targeting of GRIP1b require a PDZ ligand unique to DHHC5/8. Palmitoylated GRIP1b is targeted to trafficking endosomes and may link endosomes to kinesin motors. Consistent with this trafficking role, GRIP1b's palmitoylation turnover rate approaches the highest of all reported proteins, and palmitoylation increases GRIP1b's ability to accelerate AMPA-R recycling. To our knowledge, these findings identify the first neuronal DHHC5/8 substrate, define novel mechanisms controlling palmitoylation specificity, and suggest further links between dysregulated palmitoylation and neuropathological conditions.     

PDZ7 of glutamate receptor interacting protein binds to its target via a novel hydrophobic surface area

Glutamate receptor interacting protein 1 (GRIP1) is a scaffold protein composed of seven PDZ (Postsynaptic synaptic density-95/Discs large/Zona occludens-1) domains. The protein plays important roles in the synaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. Here, we describe the three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy. GRIP1 PDZ7 contains a closed carboxyl group-binding pocket and a narrow alphaB/betaB-groove that is not likely to bind to classical PDZ ligands. Unexpectedly, GRIP1 PDZ7 contains a large solvent-exposed hydrophobic surface at a site distinct from the conventional ligand-binding alphaB/betaB-groove. NMR titration experiments show that GRIP1 PDZ7 binds to GRASP-1 via this hydrophobic surface. Our data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins.