Biomedical subjects

Masayo Okumura

Publications and source records attributed to Masayo Okumura.

11 recordsLinked to original sources

PubMed2006

Altered localization of amyloid precursor protein under endoplasmic reticulum stress.

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.

Amyloid beta-Peptides↗

PubMed2005

Candidates for tumor-specific alternative splicing.

Gene expression can be regulated not only by transcription and post-transcriptional modifications, but also by splicing regulation. Recent genome-wide analyses have indicated that up to 70% of human genes may have alternatively spliced forms, suggesting that splicing regulation affects a wide range of gene expression. Tumor tissues show significantly altered protein expressions, and this is also thought to be affected by alternative splicing. Although some alternative splicing events have been reported to be cancer specific and others have been predicted from database analyses, the process of alternative splicing and its regulatory machinery are hardly understood. We searched for and detected alternative splicing events that alter protein splicing in all or a subset of tumor tissues. The results revealed tissue-specific alterations of splicing regulation by tumorigenesis, and regulatory cis-element analyses further suggested that multiple splicing regulatory machineries were affected by this process.

Alternative Splicing↗

PubMed2004

Tra2 beta, SF2/ASF and SRp30c modulate the function of an exonic splicing enhancer in exon 10 of tau pre-mRNA.

Some of mutations in the tau gene, which were found in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), affect alternative splicing of its exon 10 which encodes one of four microtubule-binding motifs. To examine the molecular mechanisms responsible for aberrant splicing of the tau gene containing mutations linked to FTDP-17, we performed Exon trapping and binding assay using tau exon 10 pre-mRNA and nuclear extracts of neuroblastoma cell lines and in vitro splicing using dsx-substrate. We determined that 5' site of tau exon 10 (nucleotides 12-45) possesses exonic splicing enhancer (ESE) activities in vitro splicing and the FTDP-17-linked mutations affect the ESE activities and alter the splicing patterns of tau exon 10. Tra2 beta directly and ASF/SF2 indirectly associated with the ESE of wild tau exon 10. The binding amounts of these SR proteins to tau exon 10 bearing N279K mutation increased and they enhanced splicing the mutant tau exon 10. SRp30c also enhanced the splicing of tau exon 10. These results suggest that mutations in tau exon 10 that are linked to FTDP-17 affect the ESE activities by altering the binding of some SR proteins to its pre-mRNA.

Alternative Splicing↗

PubMed2004

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions.

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.

Alternative Splicing↗

PubMed2003

Characterization of mammalian synemin, an intermediate filament protein present in all four classes of muscle cells and some neuroglial cells: co-localization and interaction with type III intermediate filament proteins and keratins.

Using a monoclonal antibody, we have detected a high molecular weight muscle protein, co-localized and co-isolating with desmin. Searching a human cDNA database with partial amino acid sequences of the protein, we found a cDNA clone encoding a 1565-amino-acid polypeptide, identified as a mammalian (human) synemin, a member of the intermediate filament (IF) protein family. Immunoblotting showed the presence of a 180-kDa polypeptide in skeletal muscle and 180- and 200-kDa polypeptides in cardiac and smooth muscles. Interestingly, synemin was also found in myoepithelial cells, which have keratin filaments instead of desmin. Moreover, synemin was also found in astrocytes of optic nerves and non-myelin-forming Schwann cells, together with glial fibrillary acidic protein (GFAP) and vimentin. Blot overlays pointed to molecular interactions of synemin with desmin, vimentin, GFAP and keratin 5 and 6, but not with keratin 14. The experimental data also suggested a possible link with nebulin, a skeletal muscle protein. Purified synemin was coassembled with desmin in different molar ratios, and at 1:25, as typically found in vivo, IFs were formed which were comparable in length to desmin filaments. However, at molar ratios of 3:25 and 6:25, much shorter and irregular shaped filamentous polymers were generated. The fact that synemin is present in all four classes of muscle cells and a specific type of glial cells is indicative of important functions. Its incorporation may give structural and functional versatility to the IF cytoskeleton.

Animals↗

PubMed2003

An intronic splicing enhancer element in survival motor neuron (SMN) pre-mRNA.

Spinal muscular atrophy is caused by the homozygous loss of survival motor neuron 1 (SMN1). SMN2, a nearly identical copy gene, differs from SMN1 only by a single nonpolymorphic C to T transition in exon 7, which leads to alteration of exon 7 splicing; SMN2 leads to exon 7 skipping and expression of a nonfunctional gene product and fails to compensate for the loss of SMN1. The exclusion of SMN exon 7 is critical for the onset of this disease. Regulation of SMN exon 7 splicing was determined by analyzing the roles of the cis-acting element in intron 7 (element 2), which we previously identified as a splicing enhancer element of SMN exon 7 containing the C to T transition. The minimum sequence essential for activation of the splicing was determined to be 24 nucleotides, and RNA structural analyses showed a stem-loop structure. Deletion of this element or disruption of the stem-loop structure resulted in a decrease in exon 7 inclusion. A gel shift assay using element 2 revealed formation of RNA-protein complexes, suggesting that the binding of the trans-acting proteins to element 2 plays a crucial role in the splicing of SMN exon 7 containing the C to T transition.

Animals↗

PubMed2003

A novel family of unconventional actins in volvocalean algae.

The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventional actin (90% amino acid identity with mammalian actin), the other a highly divergent actin (64% identity) named novel actin-like protein (NAP). To see whether the presence of conventional and unconventional actins in a single organism is unique to C. reinhardtii, we searched for genomic sequences related to the NAP sequence in several other species of volvocalean algae. Here we show that Chlamydomonas moewusii and Volvox carteri also have, in addition to a conventional actin, an unconventional actin similar to the C. reinhardtii NAP. Analyses of the deduced protein sequences indicated that the NAP homologues form a distinct group derived from conventional actin.

Actins↗

PubMed2003

MDG1/ERdj4, an ER-resident DnaJ family member, suppresses cell death induced by ER stress.

BACKGROUND: Alterations in homeostasis after various cellular stresses, which prevent protein folding and cause an accumulation of misfolding or malfolding proteins in the endoplasmic reticulum (ER), have the potential to induce cellular damage, and are therefore a type of 'ER stress.' To understand the molecular events or cascades underlying the ER stress response regulated by gene transcription and mediated by stress transducers, it is crucial to identify the molecules induced during ER stress and to analyse the roles of these genes. RESULTS: We identified MDG1/ERdj4, a member of the DnaJ protein family, as an inducible gene during ER stress. MDG1/ERdj4 contains the J domain, which is essential for interacting with Hsp70s, at the N-terminal portion and just at the back of the transmembrane domain. Its trypsin digestion and glycosylation of a chimeric protein composed of MDG1/ERdj4 fused with the extracellular domain of the amyloid precursor protein at its C-terminus, showed that its C-terminal portion containing the J domain could be orientated to the ER lumen. Over-expression of it inhibited the cell death induced by ER stress. In contrast, its mutants with the J domain deleted showed no protective effects against cell death. CONCLUSIONS: MDG1/ERdj4 may play roles in stabilizing GRP78/BiP binding to unfolded substrate proteins in a J domain-dependent manner and prevent the accumulation of unfolded proteins in the ER, consequently protecting cells from ER stress.

Amino Acid Sequence↗

PubMed2002

FAD-linked presenilin-1 mutants impede translation regulation under ER stress.

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.

Alzheimer Disease↗

PubMed2002

Identification of a cis-acting element for the regulation of SMN exon 7 splicing.

Spinal muscular atrophy results from the loss of functional survival motor neuron (SMN1) alleles. Two nearly identical copies of SMN exist and differ only by a single non-polymorphic C to T transition in exon 7. This transition leads to alteration of exon 7 splicing; that is, SMN1 produces a full-length transcript, whereas SMN2 expresses a low level of full-length transcript and predominantly an isoform lacking exon 7. The truncated transcript of SMN encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. In this paper, we identified a cis-acting element (element 1), which is composed of 45 bp in intron 6 responsible for the regulation of SMN exon 7 splicing. Mutations in element 1 or treatment with antisense oligonucleotides directed toward element 1 caused an increase in exon 7 inclusion. An approximately 33-kDa protein was demonstrated to associate with a pre-mRNA sequence containing both element 1 and the C to T transition in SMN exon 7 but not with the sequence containing mutated element 1, suggesting that the binding of the approximately 33-kDa protein plays crucial roles in the skipping of SMN exon 7 containing the C to T transition.

Alternative Splicing↗

PubMed2001

Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor).

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.

Alternative Splicing↗