TGF-ß superfamily member Nodal stimulates human ß-cell proliferation while maintaining cellular viability.

In an effort to expand human islets and enhance allogeneic islet transplant for the treatment of type 1 diabetes, identifying signaling pathways that stimulate human ß-cell proliferation is paramount. TGF-ß superfamily members, in particular activin-A, are likely involved in islet development and may contribute to ß-cell proliferation. Nodal, another TGF-ß member, is present in both embryonic and adult rodent islets. Nodal, along with its coreceptor, Cripto, are pro-proliferative factors in certain cell types. Although Nodal stimulates apoptosis of rat insulinoma cells (INS-1), Nodal and Cripto signaling have not been studied in the context of human islets. The current study investigated the effects of Nodal and Cripto on human ß-cell proliferation, differentiation, and viability. In the human pancreas and isolated human islets, we observed Nodal mRNA and protein expression, with protein expression observed in ß and α-cells. Cripto expression was absent from human islets. Furthermore, in cultured human islets, exogenous Nodal stimulated modest ß-cell proliferation and inhibited α-cell proliferation with no effect on cellular viability, apoptosis, or differentiation. Nodal stimulated the phosphorylation of mothers against decapentaplegic (SMAD)-2, with no effect on AKT or MAPK signaling, suggesting phosphorylated SMAD signaling was involved in ß-cell proliferation. Cripto had no effect on human islet cell proliferation, differentiation, or viability. In conclusion, Nodal stimulates human ß-cell proliferation while maintaining cellular viability. Nodal signaling warrants further exploration to better understand and enhance human ß-cell proliferative capacity.

Active Bax and Bak are functional holins.

The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplasm to attack the cell wall at the end of the infection cycle. We found that active Bax/Bak, but not any other Bcl-2 family protein, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Strikingly, replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Furthermore, we provide evidence that active Bax produces large membrane holes, the size of which is controlled by structural elements of Bax. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein. Together, these results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane.

Perturbation of the Bcl-2 network and an induced Noxa/Bcl-xL interaction trigger mitochondrial dysfunction after DNA damage.

How most apoptotic stimuli trigger mitochondrial dysfunction remains to be resolved. We screened the entire Bcl-2 network for its involvement in DNA damage-induced apoptosis in HeLa cells. Although the anti-apoptotic member Bcl-xL served as a major suppressor, apoptosis initiated only when both Mcl-1 and Bcl-xL were eliminated. The pro-apoptotic members Bak, Bad, Bim, and Noxa were required for apoptosis induced by DNA damaging agents camptothecin and UV. We, therefore, used a His-tagged Bcl-xL expression system to capture the relevant BH3-only proteins that bind to Bcl-xL in response to DNA damage. Surprisingly, unlike Bad and Bim, which bound Bcl-xL constitutively, Noxa became "Mcl-1-free" and interacted with Bcl-xL after DNA damage but not after death receptor engagement. Similar observations were also made in A431 cells. Importantly, this induced interaction caused cytochrome c release and apoptosis and was directly inhibited by Mcl-1, a protein eliminated or inactivated after DNA damage. These results suggest that the loss/inactivation of Mcl-1 in conjunction with an induced Noxa/Bcl-xL interaction may serve as a trigger for mitochondrial dysfunction during DNA damage-induced apoptosis.

Bax contains two functional mitochondrial targeting sequences and translocates to mitochondria in a conformational change- and homo-oligomerization-driven process.

The apoptosis gateway protein Bax normally exists in the cytosol as a globular shaped monomer composed of nine alpha-helices. During apoptosis, Bax translocates to the mitochondria, forms homo-oligomers, and subsequently induces mitochondrial damage. The mechanism of Bax mitochondrial translocation remains unclear. Among the nine alpha-helices of Bax, helices 4, 5, 6, and 9 are capable of targeting a heterologous protein to mitochondria. However, only helices 6 and 9 can independently direct the oligomerized Bax to the mitochondria. Although Bax mitochondrial translocation can still proceed with mutations in either helix 6 or helix 9, combined mutations completely abolished mitochondrial targeting in response to activating signals. Using a proline mutagenesis scanning analysis, we demonstrated that conformational changes were sufficient to cause Bax to move from the cytosol to the mitochondria. Moreover, we found that homo-oligomerization of Bax contributed to its mitochondrial translocation. These results suggest that Bax is targeted to the mitochondria through the exposure of one or both of the two functional mitochondrial targeting sequences in a conformational change-driven and homo-oligomerization-aided process.