Tissue transglutaminase is a multifunctional BH3-only protein

Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-X(L). Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity.     

A chimeric protein induces tumor cell apoptosis by delivering the human Bcl-2 family BH3-only protein Bad

Deregulation of PI3K/Akt and Raf/Mek/Erk signal transduction cascades is one of the principal causes of neoplastic transformation. The inactivation of the proapoptotic protein Bad, upon phosphorylation by different kinases of these two pathways, may play an important role in different human malignancies. Therefore, we have expressed and purified a new chimeric protein, hGM-CSF-Bad, linking the human granulocyte-macrophage colony-stimulating factor to the N-terminus of the proapoptotic protein human Bad, to deliver Bad into tumor cells and induce apoptosis. Indeed, the human GM-CSF receptor is a good target because it is overexpressed on many leukemias and solid tumors and is not detectable on stem cells. We found that the chimeric protein binds the human GM-CSF receptor, is endocytosed, and appears to reach the cytosol via retrograde ER transport. After entering cells, the protein is able to induce apoptosis of human leukemia cells and human colon and gastric carcinoma cell lines (IC(50) values as low as 1 muM). We conclude that GM-CSF-Bad can overcome the inappropriate survival stimuli in transformed cells and restore the apoptotic pathway. The completely human sequence and the elevated selectivity for cancer cells could prevent immunogenicity and the nonspecific toxicity of targeted toxins in future clinical application of this fusion protein.     

ApoL1, a BH3-only lipid-binding protein, induces autophagic cell death

We recently reported the identification and characterization of a novel BH3-only pro-death protein, apolipoprotein L1 (ApoL1), that, when overexpressed, induces autophagic cell death (ACD) in a variety of cells, including those originated from normal and cancerous tissues. ApoL1 failed to induce ACD in autophagy-deficient Atg5(-/-) and Atg7(-/-) MEF cells, suggesting that ApoL1-induced cell death is indeed autophagy-dependent. In addition, a BH3 domain deletion allele of ApoL1 was unable to induce ACD, demonstrating that ApoL1 is a bona fide BH3-only pro-death protein. To further investigate regulation of ApoL1 expression, we showed that ApoL1 is inducible by interferon-gamma and tumor necrosis factor-alpha in human umbilical vein endothelial cells, suggesting that ApoL1 may play a role in cytokine-induced inflammatory response. Moreover, we observed that ApoL1 is a lipid-binding protein with high affinity for phosphatidic acid and cardiolipin and less affinity for various phosphoinositides. Functional genomics analysis identified 5 nonsynonymous single nucleotide polymorphisms (NSNPs) in the coding exons of the human ApoL1 structural gene-all the 5 NSNPs may cause deleterious alteration of ApoL1 activity. Finally, we discuss the link between ApoL1 and various human diseases.     

唯BH3域蛋白与神经细胞凋亡

细胞凋亡在神经细胞的生理性和病理性死亡中起着重要作用。唯BH3域蛋白是Bcl-2家族中的一类仅含有BH3同源结构域的促凋亡分子,它们通过抑制Bcl-2抗凋亡成员的活性或激活Bax/Bak样促凋亡成员的活性来调节细胞凋亡。最近研究表明,唯BH3域蛋白在凋亡的启动及凋亡通路的沟通中发挥着极其重要的作用。     

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.     

Sphingosine kinase 2 inhibitor SG-12 induces apoptosis via phosphorylation by sphingosine kinase 2

Sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosine to generate sphingosine 1-phosphate, has two mammalian isotypes, SPHK1 and SPHK2. Both isozymes are promising anti-cancer therapeutic targets. In this report, we found that SG-12, a synthetic analogue of sphingosine that acts as a SPHK2 inhibitor, induces apoptosis via phosphorylation by SPHK2. The present results revealed the novel anti-cancer potential of a sphingosine analogue in the pathological setting where SPHK2 is upregulated.     

Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein

Apolipoproteins of the L family are lipid-binding proteins whose function is largely unknown. Apolipoprotein L1 and apolipoprotein L6 have been recently described as novel pro-death BH3-only proteins that are also capable of regulating autophagy. In an in-silico screening to discover novel putative BH3-only proteins, we identified yet another member of the apolipoprotein L family, apolipoprotein L2 (ApoL2), as a BH3 motif-containing protein. ApoL2 has been suggested to behave as a BH3-only protein and mediate cell death induced by interferon-gamma or viral infection. As previously described, we observed that ApoL2 protein was induced by interferon-gamma. However, knocking down its expression in HeLa cells did not regulate cell death induced by interferon-gamma. Overexpression of ApoL2 did not induce cell death on its own. ApoL2 did not sensitize or protect cells from overexpression of the BH3-only proteins Bmf or Noxa. Furthermore, siRNA against ApoL2 did not alter sensitivity to a variety of death stimuli. We could, however, detect a weak interaction between ApoL2 and Bcl-2 by immunoprecipitation of the former, suggesting a role of ApoL2 in a Bcl-2-regulated process like autophagy. However, in contrast to what has been described about its homologs ApoL1 and ApoL6, ApoL2 did not regulate autophagy. Thus, the role, if any, of ApoL2 in cell death remains to be clarified.     

The BH3-only protein bid is dispensable for DNA damage- and replicative stress-induced apoptosis or cell-cycle arrest

Bid, a caspase-activated proapoptotic BH3-only protein, is essential for Fas-induced hepatocyte destruction. Recent studies published in Cell produced conflicting results, indicating that loss of Bid either protects or enhances apoptosis induced by DNA damage or replicative stress. To resolve this controversy, we generated novel Bid-deficient mice on an inbred C57BL/6 background and removed the drug-selection cassette from the targeted locus. Nine distinct cell types from these Bid-deficient mice underwent cell-cycle arrest and apoptosis in a manner indistinguishable from control WT cells in response to DNA damage or replicative stress. Moreover, we found that even cells from the original Bid-deficient mice responded normally to these stimuli, indicating that differences in genetic background or the presence of a strong promoter within the targeted locus are unlikely to explain the differences between our results and those reported previously. We conclude that Bid has no role in DNA damage- or replicative stress-induced apoptosis or cell-cycle arrest.     

Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis

Sympathetic neuronal death induced by nerve growth factor (NGF) deprivation requires the macromolecular synthesis-dependent translocation of BAX from the cytosol to mitochondria and its subsequent integration into the mitochondrial outer membrane, followed by BAX-mediated cytochrome c (cyt c) release. The gene products triggering this process remain unknown. Here, we report that BIM, a member of the BH3-only proapoptotic subfamily of the BCL-2 protein family, is one such molecule. NGF withdrawal induced expression of BIM(EL), an integral mitochondrial membrane protein that functions upstream of (or in parallel with) the BAX/BCL-2 and caspase checkpoints. Bim deletion conferred protection against developmental and induced neuronal apoptosis in both central and peripheral populations, but only transiently, suggesting that BIM--and perhaps other BH3-only proteins--serve partially redundant functions upstream of BAX-mediated cyt c release.     

Bcl-2家族中唯BH3域蛋白的研究进展

Bcl-2家族蛋白质在细胞凋亡的调控机制中起着重要的作用,该家族包括唯BH3结构域的蛋白质（only BH3 domain protein）,如Bid、Bik、Puma、Nova、Bmf等。随着凋亡研究的深入,在哺乳动物中现已发现10多种唯BH3结构域的蛋白质,并且在凋亡中发挥重要的作用。本文主要论述唯BH3域蛋白的作用机制及其应用的研究进展。     

Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis

Sphingosine kinase-1 (SPHK1) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, sphingosine 1-phosphate, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK1. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK1 was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G1/S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK1, where SPHK1 was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.     

BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner

We report here the structure-functional characterization of a novel intronless gene, BRCC2, located on human chromosome 11q24.1. BRCC2 open reading frame (327 bp) codes for an approximately 12-kDa protein (108 amino acids (aa)) localized predominantly in the cytosol and to a lesser extent in the mitochondria. Ectopic expression of BRCC2 cDNA also was found in both the cytosol and mitochondria. Exogenous expression of BRCC2 caused apoptotic cell death in three different cell lines as evidenced by enhanced chromatin condensation, DNA fragmentation, or an enhanced number of cells in the sub-G(1) phase. In human prostate cancer cells (PC-3), BRCC2-induced DNA fragmentation was blocked efficiently by coexpression of the anti-apoptotic molecule, Bcl-X(L). Transient transfection of BRCC2 cDNA into PC-3 cells in the presence of a broad-range caspase inhibitor, Z-VAD-fmk (100 microM, 24 h), abrogated DNA fragmentation. Consistently, BRCC2 expression correlated with the activation of caspase-3 and caspase-9. An N-terminal deletion mutant of BRCC2 (10.2 kDa, Delta1-16 aa) lacking a BH3-like domain (5-12 aa, LPIEGQEI) or BRCC2 containing a mutant BH3-like domain (leucine 5-->glutamate) failed to induce apoptosis, whereas a C-terminal deletion mutant (6.8 kDa, Delta62-108 aa) retained the apoptotic activity comparable to the full-length BRCC2. Finally, the treatment of HeLa cells with doxorubicin or hydrogen peroxide (H(2)O(2)) led to an increase in the mitochondrial (heavy membrane) level of endogenous BRCC2 (doxorubicin (100 ng/ml), 5 h, approximately 2-fold; H(2)O(2) (200 microM), 2 h, approximately 2-fold). These findings demonstrate that BRCC2 functions as a proapoptotic molecule and suggest that BRCC2 induces a caspase-dependent mitochondrial pathway of cell death.