Yutaka Eguchi

Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death

Recent studies have suggested that neuronal death in Alzheimers disease or ischemia could arise from dysfunction of the endoplasmic reticulum (ER). Although caspase-12 has been implicated in ER stress-induced apoptosis and amyloid-β (Aβ)–induced apoptosis in rodents, it is controversial whether similar mechanisms operate in humans. We found that human caspase-4, a member of caspase-1 subfamily that includes caspase-12, is localized to the ER membrane, and is cleaved when cells are treated with ER stress-inducing reagents, but not with other apoptotic reagents. Cleavage of caspase-4 is not affected by overexpression of Bcl-2, which prevents signal transduction on the mitochondria, suggesting that caspase-4 is primarily activated in ER stress-induced apoptosis. Furthermore, a reduction of caspase-4 expression by small interfering RNA decreases ER stress-induced apoptosis in some cell lines, but not other ER stress-independent apoptosis. Caspase-4 is also cleaved by administration of Aβ, and Aβ-induced apoptosis is reduced by small interfering RNAs to caspase-4. Thus, caspase-4 can function as an ER stress-specific caspase in humans, and may be involved in pathogenesis of Alzheimers disease.

Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation

Apoptosis is defined by several unique morphological nuclear changes, such as chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of a family of cysteine proteases called caspases, and caspase-activated DNase (CAD/DFF40) and lamin protease (caspase-6) have been implicated in some of these changes. CAD/DFF40 induces chromatin condensation in purified nuclei, but distinct caspase-activated factor(s) may be responsible for chromatin condensation. Here we use an in vitro system to identify a new nuclear factor, designated Acinus, which induces apoptotic chromatin condensation after cleavage by caspase-3 without inducing DNA fragmentation. Immunodepletion experiments showed that Acinus is essential for apoptotic chromatin condensation in vitro, and an antisense study revealed that Acinus is also important in the induction of apoptotic chromatin condensation in cells.

Synergistic anti-apoptotic activity between Bcl-2 and SMN implicated in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron disease characterized by degeneration of the anterior horn cells of the spinal cord. It is a common fatal autosomal recessive disorder and linkage studies have identified two candidate genes, SMN (ref. 1) and NAIP (ref. 2), both on chromosome 5q13. Although NAIP protein is known to have an anti-apoptotic function, the function of SMN has been unclear and it shows no significant sequence similarity to any other protein. The SMN gene is deleted or interrupted on both chromosomes in nearly all SMA patients. Here we show that SMN interacts with Bcl-2, another anti-apoptotic protein, and that co-expression of SMN with Bcl-2 confers a synergistic preventive effect against Bax-induced or Fas-mediated apoptosis, although SMN itself has only a weak anti-apoptotic activity. SMNY272C, which carries a missense mutation and was found in an SMA patient who exceptionally retained SMN on one allele, exerts no synergism with Bcl-2. Furthermore, the product of a truncated transcript lacking exon 7, which was derived from an SMN gene carrying an intragenic mutation or from the SMN copy gene c BCD541 (ref. 1) retained in all SMA patients, had no synergistic activity but instead had a dominant-negative effect on full-length SMN. Our results indicate that an absent or decreased anti-apoptotic activity of SMN in concert with Bcl-2 underlies the pathogenesis of SMA.

A novel protein, RTN-XS, interacts with both Bcl-XL and Bcl-2 on endoplasmic reticulum and reduces their anti-apoptotic activity.

Bcl-2 and Bcl-xL serve as critical inhibitors of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. We identified two members of the reticulon (RTN) family as Bcl-xL binding proteins, i.e., NSP-C (RTN1-C) and a new family member, RTN-xS, both of which did not belong to the Bcl-2 family and were predominantly localized on the endoplasmic reticulum (ER). RTN-xS interacted with both Bcl-xL and Bcl-2, increased the localization of Bcl-xL and Bcl-2 on the ER, and reduced the anti-apoptotic activity of Bcl-xL and Bcl-2. On the other hand, NSP-C interacted only with Bcl-xL, affected the localization of Bcl-xL, and reduced Bcl-xL activity, but had no effect on Bcl-2. These results suggest that RTN family proteins can modulate the anti-apoptotic activity of Bcl-xL and Bcl-2 by binding with them and can change their localization to the ER.

Inhibition of apoptosis by the actin-regulatory protein gelsolin

Gelsolin is an actin‐regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin‐regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti‐Fas antibody, C2‐ceramide or dexamethasone, without changing the F–actin morphology or the levels of Fas or Bcl‐2 family proteins. Upon the induction of apoptosis, an increase in CPP32(‐like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro‐CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti‐Fas treatment in the gelsolintransfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(‐like) proteases strongly inhibited Fas‐mediated apoptosis, but only partially suppressed both C2‐ceramide‐ and dexamethasone‐induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(‐like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2.

SummaryCytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1Β, tumour necrosis factor-α, and interferon-γ) induced apoptotic cell death in the mouse pancreatic beta-cell line ΒTC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line αTC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in ΒTC1 cells. The abundance of endogenous Bcl-2 in ΒTC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in ΒTC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.

Complex formed by complementary RNA stem-loops and its stabilization by a protein: Function of ColE1 Rom protein

A small plasmid-specified RNA (RNA I) inhibits formation of the RNA primer for CoIE1 DNA replication by binding to its precursor (RNA II). Binding is modulated by the plasmid-specified Rom protein. Both in the presence and absence of Rom, binding starts with interaction between loops of RNAs. To understand the mechanism of binding, we examined the interactions of pairs of single stem-loops that are complementary fragments of RNA I and RNA II. We found that these complementary single stem-loops bind to each other at their loops, forming an RNAase V1-sensitive structure. Rom protects the complex from cleavage and from alkylation of phosphate groups by ethyinitrosourea. A single dimer of Rom binds to the complex by recognizing the structure rather than its exact nucleotide sequence. Rom enhances complex formation by decreasing the rate of dissociation of the complex. Structures of RNA complexes formed in the presence and absence of Rom are proposed.

Essential role of active nuclear transport in apoptosis

Background : Apoptosis is defined by chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies. Because apoptotic signals are transmitted through a common pathway that includes the target steps of death‐driving ICE‐family proteases and anti‐cell death protein Bcl‐2 in the cytoplasm, the signals must be transferred from the cytoplasm to the nucleus, at least to induce the apoptotic manifestation of the nucleus. Small signal molecules might diffuse across nuclear pores, but larger molecules are transported by active mechanisms requiring ATP and GTP hydrolysis. It is not known whether apoptotic signals are transmitted into the nucleus by the mechanisms of active nuclear transport.

Complexes formed by complementary RNA stem-loops their formations, structures and interaction with ColE1 Rom protein

Regulation of replication of plasmid ColE1 involves interaction of two plasmid-specified RNA transcripts. One of these RNAs (RNA II) serves as a primer for DNA synthesis, and the other (RNA I) is complementary to part of RNA II. The complementary regions of RNA I and RNA II form several stem-loop structures. Binding of these RNAs that regulates DNA replication begins by interaction at the loop regions. Plasmid-coded Rom protein stabilizes the product of the interaction. In this paper, the mechanism of the loop-to-loop interaction between pairs of RNA stem-loops having various nucleotide sequences is studied. Binding of two stem-loops containing six to eight nucleotides in their loops requires that the loop sequences be complementary, whereas the stem sequences need not be. The association rate constants for binding of complementary pairs with various sequences are relatively similar, around 1 x 10(6) M-1 S-1. On the other hand, the rates of dissociation of the complexes vary greatly depending on the loop sequence, even for complexes having the same base composition, suggesting a strong effect of base-stacking. All the complementary bases in the seven-nucleotide loops participate in complex formation, and the resulting complex is bent a little at the interacting region. Rom binds and stabilizes any complex formed by pairs containing fully complementary loop sequences. Structures are proposed for the RNA complexes with and without Rom.

Isolation, mapping, and functional analysis of a novel human cDNA (BNIP3L) encoding a protein homologous to human NIP3

We have isolated a novel cDNA that encodes a product showing significant sequence homology (56% identity) to human NIP3, a protein thought to interact with adenovirus E1B19kD and human BCL2 proteins. This cDNA contains an open reading frame of 657 nucleotides encoding a 219 amino acid polypeptide. The gene, designated BNIP3L, was expressed in all 16 normal human tissues examined; we mapped it to chromosome band 8p21 by fluorescence in situ hybridization. Introduction of the BNIP3L gene into six different cancer‐cell lines caused significant growth suppression in each of them, while no such effect occurred when the antisense cDNA or the vector DNA was transfected, indicating that BNIP3L may function as a tumor suppressor. Genes Chromosomes Cancer 21:230–235, 1998.