Koji Takio

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA,. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase η (ref. 4). This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase η gene. Recombinant human DNA polymerase η corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase η could be the XPV gene product.

|[alpha]|-Synuclein is phosphorylated in synucleinopathy lesions

The deposition of the abundant presynaptic brain protein α-synuclein as fibrillary aggregates in neurons or glial cells is a hallmark lesion in a subset of neurodegenerative disorders. These disorders include Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy, collectively referred to as synucleinopathies. Importantly, the identification of missense mutations in the α-synuclein gene in some pedigrees of familial PD has strongly implicated α-synuclein in the pathogenesis of PD and other synucleinopathies. However, specific post-translational modifications that underlie the aggregation of α-synuclein in affected brains have not, as yet, been identified. Here, we show by mass spectrometry analysis and studies with an antibody that specifically recognizes phospho-Ser 129 of α-synuclein, that this residue is selectively and extensively phosphorylated in synucleinopathy lesions. Furthermore, phosphorylation of α-synuclein at Ser 129 promoted fibril formation in vitro. These results highlight the importance of phosphorylation of filamentous proteins in the pathogenesis of neurodegenerative disorders.

A Serine Protease, HtrA2, Is Released from the Mitochondria and Interacts with XIAP, Inducing Cell Death

X chromosome-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspase-3, -7, and -9. Smac/DIABLO, an inhibitor of XIAP, is released from mitochondria upon receiving apoptotic stimuli and binds to the BIR2 and BIR3 domains of XIAP, thereby inhibiting its caspase-inhibitory activity. Here we report that a serine protease called HtrA2/Omi is released from mitochondria and inhibits the function of XIAP by direct binding in a similar way to Smac. Moreover, when overexpressed extramitochondrially, HtrA2 induces atypical cell death, which is neither accompanied by a significant increase in caspase activity nor inhibited by caspase inhibitors, including XIAP. A catalytically inactive mutant of HtrA2, however, does not induce cell death. In short, HtrA2 is a Smac-like inhibitor of IAP activity with a serine protease-dependent cell death-inducing activity.

Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production

Autotaxin (ATX) is a tumor cell motility–stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5′-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein–coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.

The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein

We have purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimers disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, respectively. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely associated with neuronal perikarya and dendrites. In the AD cortex, the number of GIF-positive astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.

Ubiquitin is conjugated with amino-terminally processed tau in paired helical filaments

We have investigated ubiquitinated paired helical filaments, which produce a proteinaceous smear in SDS-polyacrylamide gel electrophoresis and immunoblotting. The smear consisted largely of the carboxy-terminal portion of tau and ubiquitin. The ubiquitin-targeted protein was identified as tau in paired helical filaments, and the conjugation sites were localized to the microtubule-binding region. Most ubiquitin in paired helical filaments occurred as a monoubiquitinated form, and only a small proportion of ubiquitin formed multiubiquitin chains. There was a ubiquitin-negative smear, in which tau was much less processed in the amino-terminal portion. This strongly suggests that the amino-terminal processing of tau in paired helical filaments precedes its ubiquitination.

A Novel Motor, KIF13A, Transports Mannose-6-Phosphate Receptor to Plasma Membrane through Direct Interaction with AP-1 Complex

Intracellular transport mediated by kinesin superfamily proteins (KIFs) is a highly regulated process. The molecular mechanism of KIFs binding to their respective cargoes remains unclear. We report that KIF13A is a novel plus end-directed microtubule-dependent motor protein and associates with beta 1-adaptin, a subunit of the AP-1 adaptor complex. The cargo vesicles of KIF13A contained AP-1 and mannnose-6-phosphate receptor (M6PR). Overexpression of KIF13A resulted in mislocalization of the AP-1 and the M6PR. Functional blockade of KIF13A reduced cell surface expression of the M6PR. Thus, KIF13A transports M6PR-containing vesicles and targets the M6PR from TGN to the plasma membrane via direct interaction with the AP-1 adaptor complex.

The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex

The COP9 complex, genetically identified in Arabidopsis as a repressor of photomorphogenesis, is composed of multiple subunits including COP9, FUS6 (also known as COP11) and the Arabidopsis JAB1 homolog 1 (AJH1) ([1-3]; unpublished observations). We have previously demonstrated the existence of the mammalian counterpart of the COP9 complex and purified the complex by conventional biochemical and immunoaffinity procedures [4]. Here, we report the molecular identities of all eight subunits of the mammalian COP9 complex. We show that the COP9 complex is highly conserved between mammals and higher plants, and probably among most multicellular eukaryotes. It is not present in the single-cell eukaryote Saccharomyces cerevisiae, however. All of the subunits of the COP9 complex contain structural features that are also present in the components of the proteasome regulatory complex and the translation initiation factor eIF3 complex. Six subunits of the COP9 complex have overall similarity with six distinct non-ATPase regulatory subunits of the 26S proteasome, suggesting that the COP9 complex and the proteasome regulatory complex are closely related in their evolutionary origin. Subunits of the COP9 complex include regulators of the Jun N-terminal kinase (JNK) and c-Jun, a nuclear hormone receptor binding protein and a cell-cycle regulator. This suggests that the COP9 complex is an important cellular regulator modulating multiple signaling pathways.

Homology of perforin to the ninth component of complement (C9)

Perforin is one of the cytolytic factors present in the cytoplasmic granules of mouse cytotoxic T lymphocytes and natural killer cells1–3. We have determined the sequence of the N-terminal amino acids of perforin purified from a mouse natural killer cell line, and, by using oligonucleotide probes corresponding to the amino acid residues, we have identified a complementary DNA encoding perforin from the cDNA library of a mouse cytotoxic T lymphocyte clone. As predicted from the functional similarities between perforin and the ninth component of the serum cytolytic system, complement (C9) (refs 4–8), the deduced primary structure of perforin has homology with C9 at their respective functionally conserved regions9. We find that perforin is only expressed in killer cell lines, and not in helper T lymphocytes or other tumour cells tested. Thus we have provided direct molecular evidence that a killer-cell-specific protein evolutionally linked to C9 is involved in cell-mediated cytolysis.

Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on a partial amino acid sequence, the cDNA encoding the transferase was cloned and sequenced from a cDNA library of a human cancer cell line. The cDNA sequence has a 571-amino acid coding region indicating a protein of 64.7 kDa with a type II domain structure. The deduced protein sequence showed significant similarity to a recently cloned bovine polypeptide GalNAc-transferase (Homa, F. L., Hollanders, T., Lehman, D. J., Thomsen, D. R., and Elhammer, Å. P.(1993) J. Biol. Chem. 268, 12609-12616). A polymerase chain reaction construct was expressed in insect cells using a baculovirus vector. Northern analysis of eight human tissues differed clearly from that of the bovine GalNAc-transferase. Polymerase chain reaction cloning and sequencing of the human version of the bovine transferase are presented, and 98% similarity at the amino acid level was found. The data suggest that the purified human GalNAc-transferase is a novel member of a family of polypeptide GalNAc-transferases, and a nomenclature GalNAc-T1 and GalNAc-T2 is introduced to distinguish the members.