Glucosylation by the Legionella Effector SetA Promotes the Nuclear Localization of the Transcription Factor TFEB.

Legionella pneumophila is an intracellular pathogen that requires nutrients from the host for its replication. It has been shown that replicating L. pneumophila prefers amino acids as main sources of carbon and energy. The homeostasis of amino acids in eukaryotic cells is regulated by the transcription factor EB (TFEB), which translocates into the nucleus and activates genes for autophagy and lysosomal biogenesis. Here we show that the Legionella effector SetA causes a robust nuclear translocation of TFEB when exogenously expressed in mammalian cells and that the translocation is dependent on the glucosyltransferase activity of SetA. We further show that SetA directly glucosylates TFEB at multiple sites. Our findings of TFEB glucosylation by SetA may suggest an alternative strategy for exploiting host nutrients in addition to the control of host mTORC1 signaling by L. pneumophila. Our results provide further insight into the molecular mechanism of the delicate TFEB nuclear shuttling.

Deubiquitination of phosphoribosyl-ubiquitin conjugates by phosphodiesterase-domain-containing Legionella effectors.

Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases in the canonical ubiquitination pathway, here we show that 2 paralogous Legionella effectors, Lpg2154 (DupA; deubiquitinase for PR-ubiquitination) and Lpg2509 (DupB), reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these 2 genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella-containing vacuoles. Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.

Ordered opening of LDL receptor binding domain of human apolipoprotein E3 revealed by hydrogen/deuterium exchange mass spectrometry and fluorescence spectroscopy.

Apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in cholesterol homeostasis. The N-terminal (NT) domain of apoE3 (residues 1-191) is folded into a helix bundle comprised of 4 amphipathic α-helices: H1, H2, H3 and H4, flanked by flexible helices N1 and N2, and Hinge Helix 1 (Hinge H1), at the N-and C-terminal sides of the helix bundle, respectively. The NT domain plays a critical role in binding to the low density lipoprotein receptor (LDLR), which eventually leads to lowering of plasma cholesterol levels. In order to be recognized by the LDLR, the helix bundle has to open and undergo a conformational change. The objective of the study was to understand the mechanism of opening of the helix bundle. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed that apoE3 NT domain adopts several disordered and unfolded regions, with H2 exhibiting relatively little protection against exchange-in compared to H1, H3, and H4. Site-directed fluorescence labeling indicated that H2 not only has the highest degree of solvent exposure but also the most flexibility in the helix bundle. It also indicated that the lipoprotein behavior of H1 was significnatly different from that of H2, H3 and H4. These results suggest that the opening of the helix bundle is likely initiated at the flexible end of H2 and the loop linking H2/H3, and involves movement of H2/H3 away from H1/H4. Together, these observations offer mechanistic insight suggesting a regulated helix bundle opening of apoE3 NT domain can be triggered by lipid binding.

Charged Residues in the C-Terminal Domain of Apolipoprotein A-I Modulate Oligomerization.

Charged residues of the C-terminal domain of human apolipoprotein A-I (apoA-I) were targeted by site-directed mutagenesis. A series of mutant proteins was engineered in which lysine residues (Lys 195, 206, 208, 226, 238, and 239) or glutamate residues (Glu 234 and 235) were replaced by glutamine. The amino acid substitutions did not result in changes in secondary structure content or protein stability. Cross-linking and size-exclusion chromatography showed that the mutations resulted in reduced self-association, generating a predominantly monomeric apoA-I when five or six lysine residues were substituted. The rate of phosphatidylcholine vesicle solubilization was enhanced for all variants, with approximately a threefold rate enhancement for apoA-I lacking Lys 206, 208, 238, and 239, or Glu 234 and 235. Single or double mutations did not change the ability to protect lipolyzed low density lipoprotein from aggregation, but variants lacking >4 lysine residues were less effective in preventing lipoprotein aggregation. ApoA-I mediated cellular lipid efflux from wild-type mice macrophage foam cells was decreased for the variant with five lysine mutations. However, this protein was more effective in releasing cellular phosphatidylcholine and sphingomyelin from Abca1-null mice macrophage foam cells. This suggests that the mutations caused changes in the interaction with ABCA1 transporters and that membrane microsolubilization was primarily responsible for lipid efflux in cells lacking ABCA1. Taken together, this study indicates that ionic interactions in the C-terminal domain of apoA-I favor self-association and that monomeric apoA-I is more active in solubilizing phospholipid bilayers.

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.

Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III.

Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: role for lysine residues in antimicrobial properties.

Human apolipoprotein A-I (apoA-I) is a 28kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. In the present study the potential protective role against bacterial pathogens was explored. ApoA-I suppressed bacterial growth of Escherichia coli and Klebsiella pneumoniae. The protein was able to bind lipopolysaccharides and showed a strong preference for bilayer vesicles made of phosphatidylglycerol over phosphatidylcholine. Lysine side chains of apoA-I were acetylated to evaluate the importance of electrostatic forces in the binding interaction with both membrane components. Electrophoresis properties, dot blot analysis, circular dichroism, and fluorescence spectroscopy to probe for changes in protein structure indicated that the acetylated protein displayed a strongly reduced lipopolysaccharide and phosphatidylglycerol binding. A mutant containing only the N-terminal domain of apoA-I also showed a reduced ability to interact with the membrane components, although to a lesser extent. These results indicate the potential for apoA-I to function as an antimicrobial protein and exerts this function through lysine residues.