Non-canonical activation of the ER stress sensor ATF6 by Legionella pneumophila effectors.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) secretes ∼330 effector proteins into the host cell to sculpt an ER-derived replicative niche. We previously reported five L.p. effectors that inhibit IRE1, a key sensor of the homeostatic unfolded protein response (UPR) pathway. In this study, we discovered a subset of L.p. toxins that selectively activate the UPR sensor ATF6, resulting in its cleavage, nuclear translocation, and target gene transcription. In a deviation from the conventional model, this L.p-dependent activation of ATF6 does not require its transport to the Golgi or its cleavage by the S1P/S2P proteases. We believe that our findings highlight the unique regulatory control that L.p exerts upon the three UPR sensors and expand the repertoire of bacterial proteins that selectively perturb host homeostatic pathways

Non-canonical activation of the ER stress sensor ATF6 by Legionella pneumophila effectors.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) secretes ∼330 effector proteins into the host cell to sculpt an ER-derived replicative niche. We previously reported five L.p. effectors that inhibit IRE1, a key sensor of the homeostatic unfolded protein response (UPR) pathway. In this study, we discovered a subset of L.p. toxins that selectively activate the UPR sensor ATF6, resulting in its cleavage, nuclear translocation, and target gene transcription. In a deviation from the conventional model, this L.p-dependent activation of ATF6 does not require its transport to the Golgi or its cleavage by the S1P/S2P proteases. We believe that our findings highlight the unique regulatory control that L.p exerts upon the three UPR sensors and expand the repertoire of bacterial proteins that selectively perturb host homeostatic pathways

Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function

Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function

Characterization of chimeric apolipoproteins.

<p><b>Panel A</b>. SDS-PAGE analysis of the chimeric apolipoproteins. Electrophoresis of chimeric and parent proteins (20 μg protein) was carried out using a 4–20% acrylamide gradient Tris-glycine gel under reducing conditions in the presence of BME. <b>Panel B</b>. Western blot analysis of chimeras (0.5 μg protein) using mouse HRP-conjugated apoE polyclonal antibody (<i>Left</i>) or apoAI antibody (<i>Right</i>). Lane assignments are as follows: Lane 1, apoAI; Lanes 2, apoAI-NT/apoE-CT; Lanes 3, apoE3; Lanes 4, apoE3-NT/apoAI-CT.</p

Phospholipid vesicle solubilization capability of chimeras.

<p>About 125 μg of DMPC MLVs were equilibrated in 400 μl of PBS in a cuvette at 23.7°C in a Peltier-controlled spectrophotometer. Vesicle solubilization was initiated by addition of 125 μg of apolipoprotein, mixed rapidly and the change in absorbance at 325 nm measured for 30 min. Data were normalized to initial absorbance immediately following addition of protein. ApoAI (_____); apoAI-NT/apoE-CT (·······); apoE3 (-------); apoE3-NT/apoAI-CT (··-··-··); and DMPC vesicles alone in the absence of apolipoproteins (__ __ __).</p

Chimeric apolipoproteins mediated cholesterol efflux from J774 macrophages.

<p><b>Panels A and B.</b> J774 macrophages were labeled with [³H]cholesterol, and treated in the absence (open bars) or presence (closed bars) of cAMP. Lipid-free chimeras or parent apolipoproteins (10 μg/ml) were added to cells in serum-free RPMI-1640 medium, and amount of cholesterol efflux determined at 4 h. Conditions of time and dose were within a linear response range to allow for potential differences (if any) to be observed with and without treatment of acceptors in the absence (<b>Panel A</b>) or presence (<b>Panel B</b>) of 5x molar excess of BME. <b>Panel C.</b> Dependence of cholesterol efflux on chimera concentration. The cells were treated with the indicated concentrations of the chimeras for 4 h. <b>Panel D.</b> Kinetics of chimera-mediated cholesterol efflux. Chimera-mediated cholesterol efflux was followed at the indicated time points using 32 μg/μl of each protein. Values are means ±SD from triplicate determinations within a single experiment representative of two. For <b>Panel C</b> and <b>Panel D</b>, apoE3-NT/apoAI-CT (open inverted triangles); apoAI-NT/apoE-CT (open circles).</p

Unfolding and folding behavior of chimeras.

<p><b>Panels A and B</b>. GdnHCl-induced denaturation profiles of apoAI-NT/apoE-CT (<b>A</b>) and apoE3-NT/apoAI-CT (<b>B</b>). The samples (0.2 mg/ml) were incubated with increasing concentration of GdnHCl and 5x molar excess of TCEP for 16 h at 24°C. The ellipticity value at 222 nm was measured and protein unfolding plotted as % maximal change in which 100% represents completely unfolded protein. ApoAI (filled circles); apoAI-NT/apoE-CT (open circles); apoE3 (closed inverted triangles); and apoE3-NT/apoAI-CT (open inverted triangles). <b>Panel C</b>. ANS fluorescence emission spectra of chimeric and parent proteins. About 50 μg of each protein sample was excited at 395 nm and the emission spectra were recorded at 100 nm/min from 400 to 600 nm. ApoAI (_____); apoAI-NT/apoE-CT (·······); apoE3 (-------); apoE3-NT/apoAI-CT (··-··-··) and ANS (__ __ __).</p