Victor C. Yu

Mitochondrial translocation of cofilin is an early step in apoptosis induction

Increasing evidence suggests that movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis. Here, we report identification of the actin-binding protein cofilin by a proteomic approach, as such a factor translocated from cytosol into mitochondria after induction of apoptosis. We found that after induction of apoptosis, cofilin was translocated to mitochondria before release of cytochrome c. Reduction of cofilin protein levels with small-interfering RNA (siRNA) resulted in inhibition of both cytochrome c release and apoptosis. Only dephosphorylated cofilin was translocated to mitochondria, and the cofilin S3D mutant, which mimicks the phosphorylated form, suppressed mitochondrial translocation and apoptosis. Translocation was achieved through exposure of an amino-terminal mitochondrial targeting signal in combination with carboxy-terminal sequences. When correctly targeted to mitochondria, cofilin induced massive apoptosis. The apoptosis-inducing ability of cofilin, but not its mitochondrial localization, was dependent on the functional actin-binding domain. Thus, domains involved in mitochondrial targeting and actin binding are indispensable for its pro-apoptotic function. Our data suggest that cofilin has an important function during the initiation phase of apoptosis.

PROTEINS OF THE BCL‐2 FAMILY IN APOPTOSIS SIGNALLING: FROM MECHANISTIC INSIGHTS TO THERAPEUTIC OPPORTUNITIES

1. Proteins of the Bcl‐2 family are central regulators of apoptosis and are thought to act primarily on the mitochondria.

Structure-Activity Relationship Studies of Phenanthridine-Based Bcl-XL Inhibitors

Despite their structural similarities, the natural products chelerythrine ( 5) and sanguinarine ( 6) target different binding sites on the pro-survival Bcl-X L protein. This paper details the synthesis of phenanthridine-based analogues of the natural products that were used to probe this difference in binding profiles. The inhibitory constants for these compounds were then measured in a fluorescence polarization assay against Bcl-X L and the tagged Bak-BH3 peptide. The results led to structure-activity relationship studies, which identified the structural motifs required for binding-site specificity as well as inhibitory activity. We also identified synthetic analogues of the natural products that display similar binding modes but with more potent IC 50 values.

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine Induces Apoptosis in Human Neuroblastoma Cells, SH-SY5Y, through a p53-dependent Pathway

We have studied the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, on the regulation of apoptosis in the human neuroblastoma cell line, SH-SY5Y. H-7 (20-100 μM) induced apoptosis in these cells characterized by DNA fragmentation and chromatin condensation. Immunoblot analyses were performed with specific antibody against BCL-2, BCL-XS/L, BAX, JUNB, c-JUN, ICH-1L, c-FOS, RB, CDK-2, and p53. H-7 treatment did not significantly alter the level of these proteins with the exception of p53. H-7, but not staurosporine, caused a dramatic nuclear accumulation of p53. The kinetics of nuclear accumulation of p53 correlates well with the kinetics of induction of apoptosis. The effect of H-7 was further assessed in a group of human cell lines. Only cell lines harboring the wild-type p53 gene were responsive to the stimulatory effect of H-7 on nuclear accumulation of p53. Furthermore, cell lines carrying a mutated p53 gene were resistant to the cytotoxic effect of H-7. The ability of H-7 in mediating apoptosis in the SH-SY5Y line expressing a dominant negative mutant of p53 was significantly diminished. Taken together, these data strongly suggest that a p53-dependent mechanism contributes to the cytotoxicity of H-7 in human neuroblastoma cells.

Dynamics of RASSF1A/MOAP-1 Association with Death Receptors†

ABSTRACT RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-α stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.

A Novel BH3-like Domain in BID Is Required for Intramolecular Interaction and Autoinhibition of Pro-apoptotic Activity

Upon activation of the Fas apoptotic signaling pathway, Bid, a “BH3 domain-only” pro-apoptotic molecule, is cleaved by caspase-8 into a 6.5-kDa N-terminal and a 15-kDa BH3 domain-containing C-terminal fragment, referred to as tn-Bid and tc-Bid, respectively. tc-Bid is a more potent inducer of apoptosis than full-length Bid, suggesting that the N-terminal region of Bid has an inhibitory effect on its pro-apoptotic activity. Here, we report the identification of a novel BH3-like motif (amino acid residues 35–43) in tn-Bid. Although Bid does not homodimerize, tn-Bid is able to associate avidly with tc-Bid. Site-directed mutagenesis revealed that both the novel BH3-like and BH3 domains are necessary for direct binding between tn-Bid and tc-Bid. While full-length Bid does not associate with tn-Bid, substitution of Leu35, a critical residue in mediating tn-Bid/tc-Bid interaction, with Ala in full-length Bid is sufficient to establish Bid/tn-Bid interaction. Interestingly, the L35A Bid mutant is as effective as tc-Bid in inducing apoptosis and binding Bcl-XL. We propose that the intramolecular interaction involving the BH3-like and BH3 domains serves to regulate the pro-apoptotic potential of Bid.

Chelerythrine induces apoptosis through a Bax/Bak-independent mitochondrial mechanism.

Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (ΔΨm) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.

Structural insights into the design of small molecule inhibitors that selectively antagonize Mcl-1.

The screening of a small focused library of rhodanine derivatives as inhibitors of Bcl-2 proteins led to the discovery of two structurally related compounds with different binding profiles against the Bcl-XL and the Mcl-1 proteins. Subsequent NMR studies with mutant proteins and in silico docking studies provide a possible rationale for the observed specificity.

Inhibition of ubiquitin-mediated degradation of MOAP-1 by apoptotic stimuli promotes Bax function in mitochondria

The multidomain proapoptotic protein Bax of the Bcl-2 family is a central regulator for controlling the release of apoptogenic factors from mitochondria. Recent evidence suggests that the Bax-associating protein MOAP-1 may act as an effector for promoting Bax function in mitochondria. Here, we report that MOAP-1 protein is rapidly up-regulated by multiple apoptotic stimuli in mammalian cells. MOAP-1 is a short-lived protein (t1/2 ≈ 25 min) that is constitutively degraded by the ubiquitin-proteasome system. Induction of MOAP-1 by apoptotic stimuli ensues through inhibition of its polyubiquitination process. Elevation of MOAP-1 levels sensitizes cells to apoptotic stimuli and promotes recombinant Bax-mediated cytochrome c release from isolated mitochondria. Mitochondria depleted of short-lived proteins by cycloheximide (CHX) become resistant to Bax-mediated cytochrome c release. Remarkably, incubation of these mitochondria with in vitro-translated MOAP-1 effectively restores the cytochrome c releasing effect of recombinant Bax. We propose that apoptotic stimuli can facilitate the proapoptotic function of Bax in mitochondria through stabilization of MOAP-1.

Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interaction with the Bcl-XL Protein

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-XL, a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-XL and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-XL was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-XL. Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-XL are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-XL also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-XL at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments.