Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850 .).

The N-terminal domain of the E3L protein of vaccinia virus has sequence similarity to a family of Z-DNA binding proteins of defined three-dimensional structure and it is necessary for pathogenicity in mice. When other Z-DNA-binding domains are substituted for the similar E3L domain, the virus retains its lethality after intracranial inoculation. Mutations decreasing Z-DNA binding in the chimera correlate with decreases in viral pathogenicity, as do analogous mutations in wild-type E3L. A chimeric virus incorporating a related protein that does not bind Z-DNA is not pathogenic, but a mutation that creates Z-DNA binding makes a lethal virus. The ability to bind the Z conformation is thus essential to E3L activity. This finding may allow the design of a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L.

The vaccinia virus (VV) E3L gene is responsible for providing interferon (IFN) resistance and a broad host range to VV in cell culture. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain, which is required for the IFN resistance and broad host range of the virus, has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA-dependent protein kinase, PKR. The aminoterminal domain, while conserved among orthopoxviruses, is dispensable in cell culture. To study the role of E3L in whole-animal infections, WR strain VV recombinants either lacking E3L (VV⌬E3L) or expressing an amino-terminal (VVE3L⌬83N) or carboxy-terminal (VVE3L⌬26C) truncation of E3L were constructed. Whereas wild-type VV had a 50% lethal dose of approximately 10 4 PFU after intranasal infection, and elicited severe weight loss and morbidity, VV⌬E3L was apathogenic, leading to no death, weight loss, or morbidity. VV⌬E3L was also apathogenic after intracranial injection. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the amino-and carboxy-terminal domains of E3L were required for full pathogenesis in intranasal infections. These results demonstrate that the entire E3L gene is required for pathogenesis in the mouse model.

During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Here, we tested this hypothesis using pro-survival fulllength Bcl-x L and pro-death Bcl-x L cleavage products (⌬N61Bcl-x L and ⌬N76Bcl-x L ). Unlike Bcl-x L , ⌬N61Bcl-x L and ⌬N76Bcl-x L caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant ⌬N61Bcl-x L and ⌬N76Bcl-x L , as well as Bcl-x L , cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only ⌬N61Bcl-x L and ⌬N76Bcl-x L , but not Bcl-x L , formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-x L and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor proteinprotein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-x L cleavage. Taken together, these data are consistent with the hypothesis that Bax-like proteins oligomerize to form lipid-containing pores in the outer mitochondrial membrane, thereby releasing intermembrane apoptogenic factors into the cytosol.Proteins of the Bcl-2 family are key regulators of programmed cell death in multicellular organisms. Some members of this family, including Bax, Bak, Bok/Mtd, Bad, Bik/Nbk, Bid, Blk, Bim/Bod, and Hrk promote apoptosis, whereas others, including Bcl-2, Bcl-x L , Bcl-w, Bfl-1/A1, Mcl-1, and Boo/Diva inhibit apoptosis (1). All these proteins share one to four conserved Bcl-2 homology domains (BH) 1 designated BH1, BH2, BH3, and BH4 (1, 2). In addition, Bcl-2 family members can possess a C-terminal hydrophobic amino acid sequence that helps localize them to intracellular membranes, primarily the outer mitochondrial membrane (1, 2). The activity of Bcl-2 family proteins can be modulated not only at the transcriptional level but also by post-translational modifications (1, 3). For example, various cellular proteases have been shown to cleave Bcl-2, Bcl-x L , Bid, Bax, and Bad producing C-terminal fragments with potent pro-apoptotic activity (4 -23). Bcl-x L can be cleaved by caspase 3 after aspartate 61 and 76 and by calpain after alanine 60, converting Bcl-x L from an antiapoptotic factor to a pro-apoptotic factor (5, 6, 11). Cumulative evidence indicates that Bcl-2 relatives function, at least in part, by regulating the release of proteins enclosed in the mitochondrial intermembrane space. Current models propose that Bcl-2 family proteins exert this function either by forming pores in mitochondrial membranes themselves, or by modulating endogenous mi...

Neuronal death is often preceded by functional alterations at nerve terminals. Anti-and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons. We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells. Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast. Ntruncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL. This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL. Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions. Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC, a voltagedependent anion channel present in the outer mitochondrial membrane. These observations suggest that the mitochondrial channels specific to full-length and N-truncated BCL-xL contribute to their opposite effects on synaptic transmission, and are consistent with their opposite effects on the cell death pathway.