Noboru Sasagawa

Putative function of ADAM9, ADAM10, and ADAM17 as APP -secretase

Abstract The putative α-secretase cleaves the amyloid precursor protein (APP) of Alzheimer’s disease in the middle of the amyloid β peptide (Aβ) domain. It is generally thought that the α-secretase pathway mitigates Aβ formation in the normal brain. Several studies have suggested that ADAM9, ADAM10, and ADAM17 are candidate α-secretases belonging to the ADAM (a disintegrin and metalloprotease) family, which are membrane-anchored cell surface proteins. In this comparative study of ADAM9, ADAM10, and ADAM17, we examined the physiological role of ADAMs by expressing these ADAMs in COS-7 cells, and both “constitutive” and “regulated” α-secretase activities of these ADAMs were determined. We tried to suppress the expression of these ADAMs in human glioblastoma A172 cells, which contain large amounts of endogenous α-secretase, by lipofection of the double-stranded RNA (dsRNA) encoding each of these ADAMs. The results indicate that ADAM9, ADAM10, and ADAM17 catalyze α-secretory cleavage and therefore act as α-secretases in A172 cells. This is the first report that to suggest the endogenous α-secretase is composed of several ADAM enzymes.

A secreted form of human ADAM9 has an α-secretase activity for APP ☆

Abstract ADAM9 (MDC9, meltrin γ) is a member of the ADAM family of metalloproteases, which play important roles in cell–cell fusion, intracellular signaling, and other cellular functions. Here we cloned a novel form of human ADAM9, designated hADAM9s (s for short), which lacks the carboxyl-terminus. Human ADAM9s was found to be secreted from transfected COS cells. RT-PCR analysis demonstrated that the mRNA for hADAM9s is expressed in human brain, liver, heart, kidney, lung, and trachea. When hADAM9s was co-expressed in COS cells with APP and treated with phorbol ester, the APP was digested exclusively at the α-secretory site. These results suggest that hADAM9s has an α-secretase-like activity for APP. Non-amyloidgenic cleavage of APP may occur at the plasma membrane. Our new results support a new therapeutic strategy to decrease in the Aβ content by directly activating ADAM9 in the extracellular space.

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor.

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1‐like and D2‐like receptors. However, D2‐like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2‐like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2‐like dopamine receptors than to D1‐like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo‐lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2‐like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.

Identification of a dopamine receptor from Caenorhabditis elegans

The neurotransmitter dopamine regulates locomotion and egg laying in the nematode Caenorhabditis elegans. We have cloned a cDNA encoding the C. elegans G protein-coupled receptor (CeDOP1). The deduced amino acid sequence of the cloned cDNA shows high sequence similarities with D1-like dopamine receptors from other species. Three splice variants that differ in the length of the predicted third intracellular loop and C-terminal tail were identified. COS-7 cells transiently transfected with CeDOP1 showed high affinity binding to [(125)I]iodo-lysergic acid diethylamide (K(D)=3.43 +/- 0.83 nM). Dopamine showed the highest affinity (K(i)=0.186 microM) for this receptor among several vertebrate and invertebrate amine neurotransmitters tested, suggesting that the natural ligand for this receptor is dopamine.

BACE1 interacts with lipid raft proteins

A neuropathological hallmark of Alzheimers disease is the presence of amyloid plaques in the brain. Amyloid‐β peptide (Aβ) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by β‐ and γ‐secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Aβ generation. In support of this, APP, Aβ, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Aβ are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT‐1 binds to BACE1 (beta‐site APP cleaving enzyme 1) and that overexpression of CAV‐1 or FLOT‐1 results in recruiting BACE1 into lipid rafts and influence on β‐secretase activity in cultured cells. Our results show that both CAV‐1 and FLOT‐1 may modulate β‐secretase activity by interacting with BACE1.

MBNL1 associates with YB-1 in cytoplasmic stress granules.

The muscleblind‐like (MBNL) protein family is thought to be involved in the molecular mechanism of myotonic dystrophy (DM). Although it has been shown to have splicing activity, a broader function in cellular RNA metabolism has been implicated. In this study, we attempted to find the binding proteins of MBNL1 in order to elucidate its physiological function. First, we performed a GST pull‐down assay using GST‐MBNL1‐6xHis as bait. Several proteins were identified, including YB‐1, a multifunctional DNA/RNA‐binding protein, and DDX1, a DEAD box RNA helicase. MBNL1 formed an RNP complex with YB‐1 and DDX1 in binding assays. YB‐1 also showed a weak but significant effect on α‐actinin splice site selection. Interestingly, in response to stress, MBNL1 moved to cytoplasmic stress granules, where it colocalized with YB‐1, which was previously reported to be a component of stress granules. We found that DDX1 also colocalized with MBNL1 at stress granules. These results provide new insight into the dynamics of MBNL1 in response to stress, and they suggest a role for MBNL1 in mRNA metabolism in the cytoplasm.

Dysbindin-1, a Schizophrenia-Related Protein, Functionally Interacts with the DNA- Dependent Protein Kinase Complex in an Isoform-Dependent Manner

DTNBP1 has been recognized as a schizophrenia susceptible gene, and its protein product, dysbindin-1, is down-regulated in the brains of schizophrenic patients. However, little is known about the physiological role of dysbindin-1 in the central nervous system. We hypothesized that disruption of dysbindin-1 with unidentified proteins could contribute to pathogenesis and the symptoms of schizophrenia. GST pull-down from human neuroblastoma lysates showed an association of dysbindin-1 with the DNA-dependent protein kinase (DNA-PK) complex. The DNA-PK complex interacts only with splice isoforms A and B, but not with C. We found that isoforms A and B localized in nucleus, where the kinase complex exist, whereas the isoform C was found exclusively in cytosol. Furthermore, results of phosphorylation assay suggest that the DNA-PK complex phosphorylated dysbindin-1 isoforms A and B in cells. These observations suggest that DNA-PK regulates the dysbindin-1 isoforms A and B by phosphorylation in nucleus. Isoform C does not contain exons from 1 to 6. Since schizophrenia-related single nucleotide polymorphisms (SNPs) occur in these introns between exon 1 and exon 6, we suggest that these SNPs might affect splicing of DTNBP1, which leads to impairment of the functional interaction between dysbindin-1 and DNA-PK in schizophrenic patients.

BACE1 interacts with nicastrin.

Beta-amyloid peptide (Abeta) is generated through the proteolytic cleavage of beta-amyloid precursor protein (APP) by beta- and gamma-secretases. The beta-secretase, BACE1, initiates Abeta formation followed by gamma-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of gamma-secretase complexes, in vitro, and that nicastrin activates beta-secretase activity in COS-7 cells.

Hesr1 knockout mice exhibit behavioral alterations through the dopaminergic nervous system.

The basic helix‐loop‐helix (bHLH) transcriptional factor Hesr1 gene (hairy and enhancer of split‐related 1, also called Hey1/HRT1/CHF2/HERP2) has been identified and characterized as a member of the subfamily of hairy/Enhancer of split, and shown to be involved in cardiovascular and neural development. We report that HESR1 binds directly to a part of the 3′ non‐coding region of the human dopamine transporter (DAT1) gene and represses the endogenous DAT1 gene in HEK293 cells. To investigate functions of the HESR1 gene in the dopaminergic nervous system in vivo, we analyzed the expressions of dopamine‐related genes in the postnatal day 0 whole brains of Hesr1 knockout mice by real‐time RT‐PCR analysis. Several dopamine‐related genes, such as DAT, dopamine receptors D1, D2, D4, and D5, were significantly upregulated. Moreover, young adults of Hesr1 knockout mice showed a decrease in spontaneous locomotor activity and a reduction in exploratory behavior or behavioral responses to novelty in the open‐field, and elevated plus‐maze tests. These results indicate that the HESR1 gene is related to neuropsychiatric disorders and behavioral traits through the dopaminergic nervous system.

Interactions between homopolymeric amino acids (HPAAs)

Many human proteins contain consecutive amino acid repeats, known as homopolymeric amino acid (HPAA) tracts. Some inherited diseases are caused by proteins in which HPAAs are expanded to an excessive length. To this day, nine polyglutamine‐related diseases and nine polyalanine‐related diseases have been reported, including Huntingtons disease and oculopharyngeal muscular dystrophy. In this study, potential HPAA–HPAA interactions were examined by yeast two‐hybrid assays using HPAAs of ∼30 residues in length. The results indicate that hydrophobic HPAAs interact with themselves and with other hydrophobic HPAAs. Previously, we reported that hydrophobic HPAAs formed large aggregates in COS‐7 cells. Here, those HPAAs were shown to have significant interactions with each other, suggesting that hydrophobicity plays an important role in aggregation. Among the observed HPAA–HPAA interactions, the Ala28–Ala29 interaction was notable because polyalanine tracts of these lengths have been established to be pathogenic in several polyalanine‐related diseases. By testing several constructs of different lengths, we clarified that polyalanine self‐interacts at longer lengths (>23 residues) but not at shorter lengths (six to ∼23 residues) in a yeast two‐hybrid assay and a GST pulldown assay. This self‐interaction was found to be SDS sensitive in SDS‐PAGE and native‐PAGE assays. Moreover, the intracellular localization of these long polyalanine tracts was also observed to be disturbed. Our results suggest that long tracts of polyalanine acquire SDS‐sensitive self‐association properties, which may be a prerequisite event for their abnormal folding. The misfolding of these tracts is thought to be a common molecular aspect underlying the pathogenesis of polyalanine‐related diseases.