Abstract
Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into β-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to β-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.
Highlights
The mitochondrial pathway of apoptosis is regulated by proand anti-apoptotic members of the B-cell lymphoma 2 (BCL-2) protein family [1]
mitochondrial-derived peptide (MDP) are expressed from alternate open reading frames in regions of the mitochondrial genome that are normally transcribed for the two subunits of the mitochondrial ribosome
Amyloid fibers are incredibly stable and represent some of the most toxic, degradation-resistant complexes known to biology [58]
Summary
Fibrillation propensity between BID and HN was initially probed using light-scattering spectroscopy and CD. The titration shows that BID is initially more reactive for the fibrillation over BAX; the end points observed for titrations with either protein overlap [36]. A control titration of HN alone into 1% OG had a slightly higher baseline than usual, but there was no significant change in the incidents of scattered photons after the first titration point This demonstrates that the detergent effects on fibrillation are nonlinear. This was pushed further at pH 8.0 with an ;8-fold increase in light scattering (Fig. S2c) These observations are consistent with our hypothesis that HN targets the activated forms of proapoptotic BCL-2 proteins with more exposed hydrophobic binding sites. This is consistent with earlier reports of HN reforming into b-strands at neutral or higher pH values, and some aggregation from this phenomenon could be detected by light scattering [47, 48]
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