Humanin selectively prevents the activation of pro-apoptotic protein BID by sequestering it into fibers

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-x_L 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.

Keywords: B-cell lymphoma 2 (Bcl-2) family; BID; amyloid; apoptosis; conformational change; electron microscopy (EM); fibers; humanin; β-sheet.

Figure 1

Humanin induces conformational changes in BID, and together they form β-sheet fibers.a, increase in scattered photons from a 280-nm laser caused by aggregations in solution as a function of HN concentration in the presence (circle) or absence (square) of 5 μm BID. The error bars were calculated from the S.D. of three replicate titrations. Control titrations of buffer alone into 5 μm BID solutions did not show increased light scattering. b, CD spectra of 5 μm BID (solid line), 50 μm HN (dashed line), and a mixture of the two at the same concentrations (dotted line). The spectra were produced from three accumulated scans on a single sample. BID and HN alone produced spectra showing α-helices or disorder, respectively. Combining the two produced a spectrum with both α-helical and β-sheet properties, but there is no longer evidence of disorder, indicating that most of the HN has reformed into a stable structure. c, ThT emission spectra of the same solutions described in b. ThT did not interact with BID alone because the spectrum of the protein alone matched the buffer control, but ThT did have some activity with the peptide alone. When combined, the total fluorescence increased, and the maxima shifted by ∼7 nm, indicating formation of stable β-sheets. d, EM images of aggregations revealed fibers of uniform diameter and varying length.

Figure 2

BID is incorporated into the fiber and protected from proteolysis.a, total ion chromatograms of 2-h trypsin digestions with BID alone (top panel) and BID fibrillated with HN (bottom panel). Changes in the peak profile of BID digestion products are observed in the presence of HN. Differences in the relative intensities among three groups of peaks reveal that two regions of BID are incorporated into the fiber and are protected from proteolysis. Magenta lines highlight a long C-terminal fragment of BID, peak 8 (positions 79–202), that is more efficiently digested to a product corresponding to peak 7 (positions 126–190) when fibrillated. The peak 7 fragment spans three helices including the hydrophobic core helix α-6. Cyan lines highlight peaks 1, 3 and 4 corresponding to fragments 166–175, 133–147, and 154–164, respectively. The combination of these fragments is roughly the same sequence identified as the fragment from peak 7, which explains their attenuation in the fiber digestion. Blue lines highlight a second N-terminal region that is protected by fibrillation. Peaks 2, 5, and 6 correlating to BID fragments 43–70, 1–75, and 1–70, respectively, show that the cleavage sites in helix α-2 and a portion of the following loop region are protected from proteolysis. Peptides from HN cleavage products elute at 8.74, 24.35, 24.86, and 25.66 ml and are marked with circles. b, the sequence of BID with trypsin cleavage sites marked (asterisks) and α-helical segments highlighted. The caspase-8 cleavage sequence (6364, 65, 6667) is boxed. Some cleavage sites become more protected by the fibrillation (green asterisks), whereas others are more exposed (red asterisks).

Figure 3

Humanin mutants affect fiber morphology.a, light-scattering titration curves for 5 μm BID and each of the peptides. The WT HN/BID data (circles) are redisplayed as they appear in Fig. 1A. HN-C8A (squares) is less reactive with BID and does not form as many aggregates. HN-S14G (diamonds) aggregates less at the beginning of the titration but is within error of the WT HN values at the end. No increase in light scattering is observed with vMIA (triangles). The error bars were calculated from the S.D. of three replicate titrations. b, ThT fluorescence reported as the difference in signal intensity between solutions of 50 μm of each peptide with and without 5 μm of BAX. The HN peptides present some ThT affinity on their own but increase significantly when reacted with BID. Each fiber has a different ThT response, and the control peptide vMIA remains unchanged. The error bars were calculated from the S.D. of three replicate samples. c, EM image of fibers formed by HN C8A mutant and BID shows more irregular and thinner diameter fiber. d, EM image of HN S14G mutant and BID shows quite uniform diameter fibers that are shorter than WT HN and BID fibers.

Figure 4

Humanin does not form stable β-sheet structures with BCL-x_L.a, ThT emission spectra of samples containing 5 μm of BCL-x_L (solid lines) and with 50 μm of HN added (dotted lines). The spectrum of 50 μm HN alone (dashed lines) is shown as a control. BCL-x_L alone shows some reactivity with ThT, but when combined with HN, the two aggregate so severely that the emission spectrum only shows scattering of the 410-nm excitation beam. b, CD spectra of the samples described above using the same key. Although the combination of BID and HN resulted in enhanced signal intensity and showed evidence of β-sheet formation, combination of BCL-x_L and HN reduced signal intensity relative to free BCL-x_L along with complete loss of BCL-x_L α-helical secondary structure. BCL-x_L and HN can only form disordered globular aggregates.

Figure 5

Protected and deprotected regions of fibrillated BID. The cytosolic structure of human BID (Protein Data Bank code 2BID) highlighting regions of the protein that are protected (green) and deprotected (red) by fibrillation with HN. Helices are labeled at their N-terminal ends. A region of the p7 segment containing the caspase-8 cleavage sequence in an unstructured loop region is not protected. Also, from the p15/tBID segment, the hydrophobic core helix α6 and part of the two flanking helices comprise the largest protected segment. Parts of the tBID segment immediately N- and C-terminal to the protected region become more solvent-exposed, indicating that fibrillated BID features an extended conformation relative to the cytosolic structure. Regions colored in gray represent stretches of the amino acid sequence that transition from fiber-incorporated to solvent-exposed.