Bcl-2 and Bax interact via the BH1-3 groove-BH3 motif interface and a novel interface involving the BH4 motif.

The interaction of Bcl-2 family proteins at the mitochondrial outer membrane controls membrane permeability and thereby the apoptotic program. The anti-apoptotic protein Bcl-2 binds to the pro-apoptotic protein Bax to prevent Bax homo-oligomerization required for membrane permeabilization. Here, we used site-specific photocross-linking to map the surfaces of Bax and Bcl-2 that interact in the hetero-complex formed in a Triton X-100 micelle as a membrane surrogate. Heterodimer-specific photoadducts were detected from multiple sites in Bax and Bcl-2. Many of the interaction sites are located in the Bcl-2 homology 3 (BH3) region of Bax and the BH1-3 groove of Bcl-2 that likely form the BH3-BH1-3 groove interface. However, other interaction sites form a second interface that includes helix 6 of Bax and the BH4 region of Bcl-2. Loss-of-function mutations in the BH3 region of Bax and the BH1 region of Bcl-2 disrupted the BH3-BH1-3 interface, as expected. Surprisingly the second interface was also disrupted by these mutations. Similarly, a loss-of-function mutation in the BH4 region of Bcl-2 that forms part of the second interface also disrupted both interfaces. As expected, both kinds of mutation abolished Bcl-2-mediated inhibition of Bax oligomerization in detergent micelles. Therefore, Bcl-2 binds Bax through two interdependent interfaces to inhibit the pro-apoptotic oligomerization of Bax.

Nipissing District Developmental Screen: patterns of use by physicians in Ontario.

OBJECTIVE: To determine if providing the Nipissing District Developmental Screen (NDDS) free of charge is associated with increased use of this measure and to investigate regional variations in the use of the NDDS in Ontario. DESIGN: Retrospective analysis of purchasing data from before the NDDS was available at no cost compared with analysis of the results of a brief questionnaire completed by those downloading the NDDS for free. SETTING: Ontario. PARTICIPANTS: Users of the NDDS. INTERVENTION: Provision of free on-line access to the NDDS. MAIN OUTCOME MEASURES: Patterns of purchasing or downloading of the NDDS by FPs and health care professionals (HCPs) before and after implementation of the program. RESULTS: Before the program, 91 FPs (0.9% of FPs in Ontario) purchased the NDDS, and an additional 129 FPs (1.3% of FPs in Ontario) downloaded the NDDS in the year after the start of the program. Including all other HCPs increased the estimated number of users to 504 (representing an estimated 5.0% of all FPs in Ontario). Adjusting for group practice increased the estimate to 16.5% of all FPs in Ontario who had access to the NDDS. There were no significant differences in NDDS usage by FPs between central, southwestern, and northern Ontario (P > .05). Significantly fewer FPs in eastern Ontario accessed the NDDS than FPs in other areas of the province did (P < .001). CONCLUSION: Despite measures to increase usage, only a small number of FPs access the NDDS in Ontario. However, free access to the NDDS does seem to contribute to removing barriers to screening, as indicated by a 3-fold increase in the number of FPs accessing the NDDS. Further research is required to investigate the reasons for these trends so that effective methods to increase the use of developmental screening measures in clinical practice can be implemented.

tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation.

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.

Bcl-2 homodimerization involves two distinct binding surfaces, a topographic arrangement that provides an effective mechanism for Bcl-2 to capture activated Bax.

The homo- and heterodimerization of Bcl-2 family proteins is important for transduction and integration of apoptotic signals and control of the permeability of mitochondria and endoplasmic reticulum membranes. Here we mapped the interface of the Bcl-2 homodimer in a cell-free system using site-specific photocross-linking. Bcl-2 homodimer-specific photoadducts were detected from 11 of 17 sites studied. When modeled into the structure of Bcl-2 core, the interface is composed of two distinct surfaces: an acceptor surface that includes the hydrophobic groove made by helices 2 and 8 and the loop connecting helices 4 and 5 and a donor surface that is made by helices 1-4 and the loop connecting helices 2 and 3. The two binding surfaces are on separate faces of the three-dimensional structure, explaining the formation of Bcl-2 homodimers, homo-oligomers, and Bcl-2/Bax hetero-oligomers. We show that in vitro the Bcl-2 dimer can still interact with activated Bax as a larger oligomer. However, formation of a Bax/Bcl-2 heterodimer is favored, since this interaction inhibits Bcl-2 homodimerization. Our data support a simple model mechanism by which Bcl-2 interacts with activated Bax during apoptosis in an effective manner to neutralize the proapoptotic activity of Bax.