Bernard A. Callus

TRAF2 Must Bind to Cellular Inhibitors of Apoptosis for Tumor Necrosis Factor (TNF) to Efficiently Activate NF-κB and to Prevent TNF-induced Apoptosis

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-κB-inducing kinase stability and suppress constitutive noncanonical NF-κB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-κB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-κB signaling and that efficient activation of NF-κB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.

Caspase inhibitors: viral, cellular and chemical

Caspases, key mediators of apoptosis, are a structurally related family of cysteine proteases that cleave their substrates at aspartic acid residues either to cause cell death or to activate cytokines as part of an immune response. They can be controlled upstream by the regulation of signals that lead to zymogen activation, or downstream by inhibitors that prevent them from reaching their substrates. This review specifically looks at caspase inhibitors as distinct from caspase regulators: those produced by the cell itself; those whose genes are carried by viruses; and artificial caspase inhibitors used for research and potentially as therapeutics.

Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation.

Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro‐apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C‐terminal coiled‐coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled‐coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N‐terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C‐terminal coiled‐coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.

SOCS36E, a novel Drosophila SOCS protein, suppresses JAK/STAT and EGF-R signalling in the imaginal wing disc

We have cloned a novel SOCS gene from Drosophila, socs36E, which is most homologous to the mammalian socs-5 gene. Socs36E is expressed zygotically, predominantly during embryogenesis, in a highly dynamic pattern. In vivo expression of SOCS36E in transgenic flies results in several adult phenotypes. Engrailed-GAL4 directed expression causes loss of the wing anterior cross vein, humeral outgrowths, absence of halteres and eye pigmentation defects. Expression of SOCS36E under apterous-GAL4 control resulted in outstretched wings. Full penetrance of these phenotypes required the presence of the SH2 and SOCS-box domains of SOCS36E. The observed phenotypes were consistent with defects in JAK/STAT or EGF-R signalling and were exacerbated in flies heterozygous for either the d-jak (hopscotch), d-stat (stat92E) or d-egf-r (der) genes. Conversely, inactivating one copy of the d-cbl gene, a negative regulator of the d-EGF-R, partially rescued the wing phenotypes. These genetic interactions imply that SOCS36E can suppress activities of the JAK/STAT and EGF-R signalling pathways in the wing disc and suggest that SOCS36E interacts with multiple pathways in vivo.

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma−/− cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-xL and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

Bioenergetic differences selectively sensitize tumorigenic liver progenitor cells to a new gold(I) compound.

A hallmark of cancer cells is their ability to evade apoptosis and mitochondria play a critical role in this process. Delineating mitochondrial differences between normal and cancer cells has proven challenging due to the lack of matched cell lines. Here, we compare two matched liver progenitor cell (LPC) lines, one non-tumorigenic [p53-immortalized liver (PIL) 4] and the other tumorigenic (PIL2). Analysis of these cell lines and a p53 wild-type non-tumorigenic cell line [bipotential murine oval liver (BMOL)] revealed an increase in expression of genes encoding the antiapoptotic proteins cellular inhibitor of apoptosis protein (cIAP) 1 and yes associate protein in the PIL2 cells, which resulted in an increase in the protein encoded by these genes. PIL2 cells have higher mitochondrial membrane potential (Deltapsi(m)) compared with PIL4 and BMOL and had greater levels of reactive oxygen species, despite the fact that the mitochondrial antioxidant enzyme, manganese superoxide disumutase, was elevated at transcript and protein levels. Taken together, these results may account for the observed resistance of PIL2 cells to apoptotic stimuli compared with PIL4. We tested a new gold compound to show that hyperpolarized Deltapsi(m) led to its increased accumulation in mitochondria of PIL2 cells. This compound selectively induces apoptosis in PIL2 cells but not in PIL4 or BMOL. The gold compound depolarized the Deltapsi(m), depleted the adenosine triphosphate pool and activated caspase-3 and caspase-9, suggesting that apoptosis was mediated via mitochondria. This investigation shows that the non-tumorigenic and tumorigenic LPCs are useful models to delineate the role of mitochondrial dysfunction in tumorigenesis and for the future development of mitochondria-targeted chemotherapeutics that selectively target tumor cells.

Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.

Distinct requirements for the Sprouty domain for functional activity of Spred proteins

Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G(0)-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.

Regulation of microRNAs and their role in liver development, regeneration and disease.

Since their discovery more than a decade ago microRNAs have been demonstrated to have profound effects on almost every aspect of biology. Numerous studies in recent years have shown that microRNAs have important roles in development and in the etiology and progression of disease. This review is focused on microRNAs and the roles they play in liver development, regeneration and liver disease; particularly chronic liver diseases such as alcoholic liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, viral hepatitis and primary liver cancer. The key microRNAs identified in liver development and chronic liver disease will be discussed together with, where possible, the target messenger RNAs that these microRNAs regulate to profoundly alter these processes. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.

Triggering of apoptosis by Puma is determined by the threshold set by prosurvival Bcl-2 family proteins.

Puma (p53 upregulated modulator of apoptosis) belongs to the BH3 (Bcl-2 homology 3)-only protein family of apoptotic regulators. Its expression is induced by various apoptotic stimuli, including irradiation and cytokine withdrawal. Using an inducible system to express Puma, we investigated the nature of Puma-induced apoptosis. In BaF(3) cells, expression of Puma caused rapid caspase-mediated cleavage of ICAD (inhibitor of caspase-activated deoxyribonuclease) and Mcl-1 (myeloid cell leukemia 1), leading to complete loss of cell viability. Surprisingly, Puma protein levels peaked within 2 h of its induction and subsequently declined to basal levels. Maximal Puma abundance coincided with the onset of caspase activity. Subsequent loss of Puma was prevented by the inhibition of caspases, indicating that its degradation was caspase dependent. In cells expressing transfected Bcl-2, induced Puma reached significantly higher levels, but after a delay, caspases became active and cell death occurred. Puma co-immunoprecipitated endogenous Bcl-2 and Mcl-1 but not Bax and Bak, suggesting that Puma did not associate with either Bax or Bak in these cells to initiate cell death. In mouse embryonic fibroblasts (MEFs), the amount of Puma peaked within 4 h of its induction. In contrast, in bax/bak double-knockout MEFs, Puma was stably expressed following its induction and was unable to trigger apoptosis even at very high levels. Overexpression of Bcl-2 in wild-type MEFs, like in BaF(3) cells, resulted in higher levels of Puma being reached but did not prevent cell death from occurring. These results demonstrate that the level of the Bcl-2 prosurvival family sets the threshold at which Puma is able to indirectly activate Bax or Bak, leading in turn to activation of caspases that not only cause cell death but also rapidly induce Puma degradation.