Isolation of Bcl-2 binding proteins that exhibit homology with BAG-1 and suppressor of death domains protein.

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis and is overexpressed in a variety of different malignancies. Bcl-2 function is regulated through heterodimerization with other members of the Bcl-2 protein family. In addition, several proteins that are not members of the Bcl-2 family can bind to Bcl-2, including BAG-1 protein. In this study, we screened for proteins that bind to Bcl-2, and isolated two additional members of the BAG-1 protein family, BAG-3 and BAG-4. The BAG-4 protein that we cloned also corresponds to the recently isolated suppressor of death domains (SODD) protein, a molecule that binds and inhibits signaling by tumor necrosis factor receptor 1 (TNFR1). Both BAG-3 and BAG-4/SODD were found to physically associate with Bcl-2, and both proteins are well conserved from human to mouse. A region of homology, comprising 68 amino acids, is present in the carboxyl termini of BAG-3 and BAG-4/SODD, and this region corresponds with sequences termed BAG domains that are found in other members of the BAG-1 protein family. In BAG-3 and BAG-4/SODD, the BAG domains appear to constitute the Bcl-2 binding regions of these molecules. BAG-3 and BAG-4/SODD, like BAG-1, were also shown to bind to Hsp70 inside the cell. Moreover, BAG-3 overexpression modestly inhibited apoptosis resulting from cytokine deprivation of IL-3-dependent 32D cells. Together, our findings demonstrate that other members of the BAG-1 protein family, namely BAG-3 and BAG-4/SODD, bind to Bcl-2 and provide a potential link between pathways regulated by Bcl-2 and pathways regulated by Hsp70, as well as TNFR1.

Altered immune responses to a heterologous protein in ponies with heavy gastrointestinal parasite burdens.

This study was performed to test the hypothesis that immunity to heterologous vaccination would improve when the parasites were removed. It was also expected that parasitised ponies would exhibit a biased Th2 cytokine response to KLH immunisation. Helminth parasites are common in horses even in the era of highly effective broad-spectrum antiparasiticides. These parasites have been shown to alter the outcome to heterologous immunisation in a number of host species. The effect of gastrointestinal parasites on heterologous vaccination has not been addressed in equids. In the current study, humoral, lymphoproliferative, and cytokine responses to a single i.m. injection of keyhole limpet haemocyanin (KLH) were compared between groups of ponies with high, medium or low gastrointestinal parasite burdens. Antibody levels determined by ELISA showed that animals with low levels of parasites had a trend toward increased KLH specific total immunoglobulin, IgG(T) and IgA compared to heavily parasitised ponies. Medium and heavily parasitised ponies demonstrated a trend toward reduced lymphoproliferative response to KLH that was not restored after the addition of interleukin-2 (Il-2). Cells from these ponies also produced significantly lower levels of IL-4 compared to lightly parasitised ponies. These data indicate that heavily parasitised ponies have uniformly decreased cellular and humoral immune responses to soluble protein immunisation. The mechanisms involved may have potential deleterious effects on standard vaccine protocols of parasitised equines.

Analysis of the role of conserved cysteine residues in the bcl-2 oncoprotein.

The Bcl-2 oncoprotein is an integral membrane protein localized primarily to the outer membrane of the mitochondria. The precise molecular mechanism responsible for the antiapoptotic action of Bcl-2 remains unknown. Two cysteine residues are found in Bcl-2 and these residues are well-conserved across species. The first cysteine (cys(155)) is located in the alpha5 domain, a region important for the ion channel properties of Bcl-2, while the second cysteine (cys(226)) is located in the carboxyl-terminal membrane anchor domain. In this study, we found that replacement of both cysteines with serine residues generated a mutant protein that retained the ability to homodimerize and heterodimerize with proapoptotic Bax protein in vitro. In whole cells, the mutant protein efficiently heterodimerized with Bax, but exhibited impaired homodimerizationrelative to wild-type Bcl-2. The mutant protein was also less efficient than wild-type Bcl-2 at suppressing caspase activation, DNA fragmentation, and loss of viability during IL-3 withdrawal-induced apoptosis. Together, the data indicate that the cysteine residues in Bcl-2 contribute, but are not absolutely essential, to the ability of Bcl-2 to homodimerize, heterodimerize with Bax, and suppress apoptosis.

Bcl-2- and CrmA-inhibitable dephosphorylation and cleavage of retinoblastoma protein during etoposide-induced apoptosis.

Cell numbers are regulated by a balance between proliferation and apoptosis (programmed cell death). Recent evidence suggests that proteins regulating cell proliferation also mediate apoptosis. Therefore, cellular fate might be determined by cross talk between regulators of cell cycle progression and apoptosis. Previously, we had found that during DNA damage-induced apoptosis, retinoblastoma protein (RB), an important G1/S regulator and tumor suppressor, became dephosphorylated and then immediately cleaved into p48 and p68 fragments. Here, we report that expression of the Bcl-2 oncoprotein, an inhibitor of caspases (interleukin 1 -converting enzyme-like proteases), blocked RB dephosphorylation, RB cleavage and apoptosis in etoposide-treated human Jurkat T cells. In addition, expression of the cowpox virus CrmA protein, a direct inhibitor of caspases, also inhibited both RB changes and apoptosis. Taken together, our findings demonstrate important roles for caspases in the processes of etoposide-induced RB dephosphorylation, RB proteolysis and apoptosis.

IL-3 withdrawal activates a CrmA-insensitive poly(ADP-ribose) polymerase cleavage enzyme in factor-dependent myeloid progenitor cells.

Murine myeloid progenitor cells that are dependent on interleukin-3 (IL-3) undergo apoptosis when this essential cytokine is withdrawn. To determine whether IL-3 withdrawal leads to the activation of caspase proteases, known mediators of apoptosis, we studied proteolytic cleavage of the caspase substrate protein poly(ADP-ribose) polymerase (PARP) in two IL3-dependent myeloid progenitor cell lines, 32D and FDCP-1. We observed that IL-3 withdrawal leads to PARP cleavage in both cell lines, with complete cleavage occurring by 24 h after cytokine removal. The induced PARP cleavage activities were blocked by the caspase inhibitors z-DEVD-fluoromethyl ketone (z-DEVD-FMK) and z-VAD-fluoromethyl ketone (z-VAD-FMK), or by overexpression in 32D cells of Bcl-2 or BCR/ABL. By contrast, overexpression in 32D cells of cowpox virus CrmA protein, an inhibitor of Fas-mediated PARP cleavage, failed to inhibit PARP cleavage following IL-3 withdrawal. CrmA also failed to block DNA fragmentation and loss of cell viability. We propose that a CrmA-insensitive caspase protease is activated in the IL-3-deprived myeloid precursors, and that activation of this protease may direct the cells on a path towards commitment to death.

Inhibition of caspase proteases by CrmA enhances the resistance of human leukemic cells to multiple chemotherapeutic agents.

Proteases that are members of the caspase (or interleukin-1beta converting enzyme (ICE)) protease family have been shown to be important mediators of apoptosis induced by Fas activation, neurotrophic factor withdrawal, and detachment from extracellular matrix. In this report we have investigated the potential importance of caspase proteases in apoptosis induced by multiple chemotherapeutic agents. Human T leukemic cells engineered to overexpress the cowpox virus CrmA protein, a direct and specific inhibitor of caspase proteases, were studied for their resistance to 1-beta-D-arabinofurasosyl-cytosine (Ara-C), etoposide (VP-16), doxorubicin (DOX), and cis-dichlorodiammine platinum (CP). Overexpression of CrmA dramatically inhibited drug-induced activation of caspases, as measured by processing of the inactive precursor form of caspase-3 and cleavage of caspase substrate proteins poly(ADP-ribose) polymerase (PARP) and lamin B. CrmA also significantly inhibited the kinetics of cell death induced by each of the four drugs. Moreover, when examined several days or weeks after initial exposure to drug, cultures of CrmA-expressing cells were found to have recovered and repopulated, whereas vector-transfected control cells did not. These studies demonstrate that caspase proteases play an important role in conferring sensitivity to multiple chemotherapy drugs, and that constitutive downmodulation of caspase activities can enhance chemoresistance.

Fas stimulation induces RB dephosphorylation and proteolysis that is blocked by inhibitors of the ICE protease family.

Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family. Stimulation of Fas by Fas ligand or agonistic antibodies results in the activation of interleukin-1 beta converting enzyme-like (ICE-like) proteases, and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Ultimately, Fas activation leads to apoptotic cell death. The importance of PARP cleavage to the death process remains unclear. We have hypothesized that the cleavage of other cellular substrates may be important for Fas-mediated apoptosis. Here we show that stimulation of Fas results in significant alterations of retinoblastoma protein (RB). Treatment of Jurkat cells, a human leukemic T cell line, with anti-Fas induces dephosphorylation of RB, followed by proteolytic cleavage. These events precede internucleosomal DNA fragmentation. Dephosphorylation and cleavage of RB are inhibited by a specific tetrapeptide inhibitor of ICE-like proteases or by expression of cowpox virus CrmA protein or the Bcl-2 oncoprotein. Inhibition of these RB changes correlates with inhibition of apoptosis. We propose that cleavage of RB may represent an important step in the pathway of Fas-mediated apoptotic cell death.