Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells.
ABSTRACT: Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an alpha-beta hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V(max) than the full-length form without changes in K(m), which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic alpha-helix (bold) within amino acid residues 10-24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic alpha-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.
Project description:Within tuberculous granulomas, a subpopulation of Mycobacterium tuberculosis resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with M. tuberculosis-containing phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain to TAG hydrolysis remains unclear. Here, enzymatic studies were performed to compare the lipolytic activities of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(ΔPE). Complementarily, an FM model was used where bone marrow-derived mouse macrophages were infected with M. bovis BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a lipY deletion mutation prior to being exposed to TAG-rich very-low-density lipoprotein (VLDL). Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI (ILI+3) were not formed because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases, (ii) ILI+3 profiles in the strain overexpressing LipY(ΔPE) were reduced, and (iii) the number of ILI+3 profiles in the ΔlipY mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.
Project description:While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes, including UCP-1 with lower PPAR? binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining a BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.
Project description:Streptococcus mutans secretes and utilizes a 21-amino-acid signaling peptide pheromone to initiate quorum sensing for genetic competence, biofilm formation, stress responses, and bacteriocin production. In this study, we designed and synthesized a series of truncated peptides and peptides with amino acid substitutions to investigate their structure-activity relationships based on the three-dimensional structures of S. mutans wild-type signaling peptide UA159sp and C-terminally truncated peptide TPC3 from mutant JH1005 defective in genetic competence. By analyzing these peptides, we demonstrated that the signaling peptide of S. mutans has at least two functional domains. The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activation of the signal transduction pathway, while the core alpha-helical structure extending from residue 5 to the end of the peptide is required for receptor binding. Peptides in which three or more residues were deleted from the C terminus did not induce genetic competence but competitively inhibited quorum sensing activated by UA159sp. Disruption of the amphipathic alpha-helix by replacing the Phe-7, Phe-11, or Phe-15 residue with a hydrophilic residue resulted in a significant reduction in or complete loss of the activity of the peptide. In contrast to the C-terminally truncated peptides, these peptides with amino acid substitutions did not compete with UA159sp to activate quorum sensing, suggesting that disruption of the hydrophobic face of the alpha-helical structure results in a peptide that is not able to bind to the receptor. This study is the first study to recognize the importance of the signaling peptide C-terminal residues in streptococcal quorum sensing.
Project description:Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.
Project description:The group B streptococcus type I pullulanase (SAP) is a class 13 glycoside hydrolase that is anchored to the bacterial cell surface via a conserved C-terminal anchoring motif and involved in alpha-glucan degradation. Recent in vitro functional studies have shown that SAP is immunogenic in humans and that anti-SAP sera derived from immunized animals impair both group A and group B streptococcus pullulanase activities, suggesting that in vivo immunization with this antigen could prevent streptococcal colonization. To further investigate the putative role of SAP in bacterial pathogenesis, we carried out functional studies and found that recombinant SAP binds to human cervical epithelial cells. Furthermore, with a view of using SAP as a vaccine candidate, we present high-resolution crystal structure analyses of an N-terminally truncated form of SAP lacking the carbohydrate binding module but containing the catalytic domain and displaying glycosidase hydrolase activity, both in its apo form and in complex with maltotetraose, at resolutions of 2.1 and 2.4 A, respectively.
Project description:Lipid droplets are intracellular lipid-storage organelles that are thought to be derived from the endoplasmic reticulum (ER). Several pathogens, notably hepatitis C virus, use lipid droplets for replication. Numerous questions remain about how lipid droplets are generated and used by viruses. Here we show that the IFN-induced antiviral protein viperin, which localizes to the cytosolic face of the ER and inhibits HCV, localizes to lipid droplets. We show that the N-terminal amphipathic alpha-helix of viperin that is responsible for ER localization is also necessary and sufficient to localize both viperin and the fluorescent protein dsRed to lipid droplets. Point mutations in the alpha-helix that prevent ER association also disrupt lipid droplet association, and sequential deletion mutants indicate that the same number of helical turns are necessary for ER and lipid droplet association. Finally, we show that the N-terminal amphipathic alpha-helix of the hepatitis C viral protein NS5A can localize dsRed and viperin to lipid droplets. These findings indicate that the amphipathic alpha-helices of viperin and NS5A are lipid droplet-targeting domains and suggest that viperin inhibits HCV by localizing to lipid droplets using a domain and mechanism similar to that used by HCV itself.
Project description:Excessive caloric intake leading to obesity is associated with insulin resistance and dysfunction of islet ? cells. High-fat feeding decreases desnutrin (also called ATGL/PNPLA2) levels in islets. Here we show that desnutrin ablation via RIP-Cre (?KO) or RIP-CreER results in hyperglycemia with impaired glucose-stimulated insulin secretion (GSIS). Due to decreased lipolysis, islets have higher TAG content but lower free FA levels. ?KO islets exhibit impaired mitochondrial respiration and lower production of ATP required for GSIS, along with decreased expression of PPAR? target genes involved in mitochondrial oxidation. Furthermore, synthetic PPAR?, but not PPAR?, agonist restores GSIS and expression of mitochondrial oxidative genes in ?KO mice, revealing that desnutrin-catalyzed lipolysis generates PPAR? ligands. Finally, adenoviral expression of desnutrin in ?KO islets restores all defects of ?KO islet phenotype and function, including GSIS and mitochondrial defects, demonstrating the critical role of the desnutrin-PPAR?-mitochondrial oxidation axis in regulating islet ? cell GSIS.
Project description:Apolipoprotein A-I is the major protein in high-density lipoprotein (HDL) and plays an important role during the process of reverse cholesterol transport (RCT). Knowledge of the high-resolution structure of full-length apoA-I is vital for a molecular understanding of the function of HDL at the various steps of the RCT pathway. Due to the flexible nature of apoA-I and aggregation properties, the structure of full-length lipid-free apoA-I has evaded description for over three decades. Sequence analysis of apoA-I suggested that the amphipathic ?-helix is the structural motif of exchangeable apolipoprotein, and NMR, X-ray and MD simulation studies have confirmed this. Different laboratories have used different methods to probe the secondary structure distribution and organization of both the lipid-free and lipid-bound apoA-I structure. Mutation analysis, synthetic peptide models, surface chemistry and crystal structures have converged on the lipid-free apoA-I domain structure and function: the N-terminal domain [1-184] forms a helix bundle while the C-terminal domain [185-243] mostly lacks defined structure and is responsible for initiating lipid-binding, aggregation and is also involved in cholesterol efflux. The first 43 residues of apoA-I are essential to stabilize the lipid-free structure. In addition, the crystal structure of C-terminally truncated apoA-I suggests a monomer-dimer conversation mechanism mediated through helix 5 reorganization and dimerization during the formation of HDL. Based on previous research, we have proposed a structural model for full-length monomeric apoA-I in solution and updated the HDL formation mechanism through three states. Mapping the known natural mutations on the full-length monomeric apoA-I model provides insight into atherosclerosis development through disruption of the N-terminal helix bundle or deletion of the C-terminal lipid-binding domain.
Project description:tmRNA is a bacterial small RNA having a structure resembling the upper half of tRNA and its 3' end accepts alanine followed by binding to EF-Tu like tRNA. Instead of lacking a lower half of the cloverleaf structure including the anticodon, tmRNA has a short coding sequence for tag-peptide that serves as a target of cellular proteases. An elaborate coordination of two functions as tRNA and mRNA facilitates an irregular translation termed trans-translation: a single polypeptide is synthesized from two mRNA molecules. It allows resumption of translation stalled on a truncated mRNA, producing a chimeric polypeptide comprising the C-terminally truncated polypeptide derived from truncated mRNA and the C-terminal tag-peptide encoded by tmRNA. Trans-translation promotes recycling of the stalled ribosomes in the cell, and the resulting C-terminally tagged polypeptide is preferentially degraded by cellular proteases. Biochemical studies using in vitro trans-translation systems together with structural studies have unveiled the molecular mechanism of trans-translation, during which the upper and lower halves of tRNA are mimicked by the tRNA-like structure of tmRNA and a tmRNA-specific binding protein called SmpB, respectively. They mimic not only the tRNA structure but also its behavior perhaps at every step of the trans-translation process in the ribosome. Furthermore, the C-terminal tail of SmpB, which is unstructured in solution, occupies the mRNA path in the ribosome to play a crucial role in trans-translation, addressing how tmRNA·SmpB recognizes the ribosome stalled on a truncated mRNA.
Project description:Triacylglycerol (TAG) hydrolysis, membrane lipid biosynthesis, and lipid turnover are largely interlinked processes. In yeast, TAG is mobilized by three TAG lipases named Tgl3p, Tgl4p, and Tgl5p, which are localized to lipid particles/droplets. These TAG lipases posses a conserved GXSXG motif that is characteristic of hydrolytic enzymes. Here, we demonstrated that the yeast TAG lipase Tgl4p, the functional ortholog of the adipose TAG lipase, ATGL, catalyzes multiple functions in lipid metabolism. An extended domain and motif search analysis revealed that Tgl4p bears not only a lipase consensus domain but also a conserved motif for calcium-independent phospholipase A(2). We show that Tgl4p exhibits TAG lipase, steryl ester hydrolase, and phospholipase A(2) activities, but surprisingly it also catalyzed the acyl-CoA-dependent acylation of lysophosphatidic acid to phosphatidic acid (PA). Heterologous overexpression of Tgl4p in Pichia pastoris increased total phospholipid and specifically PA synthesis. Moreover, deletion of TGL4 in Saccharomyces cerevisiae showed an altered pattern of phosphatidylcholine and PA molecular species. Altogether, our data suggest that yeast Tgl4p functions as a hydrolytic enzyme in lipid degradation but also contributes to fatty acid channeling and phospholipid remodeling.