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1.
Cell Microbiol ; 20(2)2018 02.
Article in English | MEDLINE | ID: mdl-29117636

ABSTRACT

Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild-type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.


Subject(s)
Chlamydia trachomatis/physiology , Fatty Acids/metabolism , Growth and Development/physiology , Host-Pathogen Interactions/physiology , Lipid Droplets/metabolism , Cell Line, Tumor , Chlamydia trachomatis/metabolism , HeLa Cells , Humans , Inclusion Bodies/metabolism , Inclusion Bodies/physiology , Oxygen Consumption/physiology , Phospholipids/metabolism , Vacuoles/metabolism , Vacuoles/physiology
2.
Sci Rep ; 6: 23148, 2016 Mar 18.
Article in English | MEDLINE | ID: mdl-26988341

ABSTRACT

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.


Subject(s)
Anti-Bacterial Agents/pharmacology , Chlamydia trachomatis/physiology , Coenzyme A Ligases/metabolism , Vacuoles/metabolism , Chlamydia trachomatis/drug effects , Chlamydia trachomatis/growth & development , Coenzyme A Ligases/antagonists & inhibitors , Dose-Response Relationship, Drug , Fatty Acids/metabolism , HeLa Cells , Hep G2 Cells , Host-Pathogen Interactions , Humans , Protein Transport/drug effects
3.
Nucleic Acids Res ; 39(18): 8187-99, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21715379

ABSTRACT

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.


Subject(s)
DNA Polymerase I/chemistry , DNA Polymerase I/ultrastructure , DNA Primase/chemistry , DNA Primase/ultrastructure , Amino Acid Sequence , DNA Polymerase I/metabolism , DNA Primase/metabolism , Models, Molecular , Molecular Sequence Data , Protein Subunits/chemistry , RNA/chemistry , Saccharomyces cerevisiae/enzymology
4.
DNA Repair (Amst) ; 8(12): 1380-9, 2009 Dec 03.
Article in English | MEDLINE | ID: mdl-19837014

ABSTRACT

The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of approximately 37A reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.


Subject(s)
DNA Ligases/metabolism , DNA Ligases/ultrastructure , DNA Repair , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/ultrastructure , DNA/metabolism , DNA Ligase ATP , DNA Ligases/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Humans , Microscopy, Electron , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary
5.
Biochim Biophys Acta ; 1794(8): 1211-7, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19409513

ABSTRACT

Syk is a cytoplasmic tyrosine kinase that is activated after recruitment to immune receptors, triggering the phopshorylation of downstream targets. The kinase activity of Syk is controlled by an auto-inhibited conformation consisting of a regulatory region that contains two N-terminal Src homology 2 (SH2) domains inhibiting the catalytic activity of the kinase domain located at the C-terminus. The atomic structure of the related Zap-70 kinase and an electron microscopy (EM) model of Syk have revealed the structural mechanism of this auto-inhibition based on the formation of a compact conformation sustained by interactions between the regulatory and catalytic domains. On the other hand, the structural basis of Syk activation is not fully understood due to the lack of a 3D structure of full-length Syk in an active conformation. Here, we have used single-particle electron microscopy to analyse the conformational changes taking place in an activated form of Syk induced by auto-phosphorylation. The conformation of phosphorylated Syk is reminiscent of the compact structure of the inhibited protein but significant conformational changes are observed in the regulatory region. These rearrangements could be sufficient to disrupt the inhibitory interactions, contributing to Syk activation. These results suggest that the regulation of the activation of Syk might be modulated by subtle changes in the positioning of the regulatory domains rather than a full opening mechanism as proposed for the Src kinases.


Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Protein-Tyrosine Kinases/chemistry , Animals , Enzyme Activation , Intracellular Signaling Peptides and Proteins/metabolism , Microscopy, Electron , Models, Molecular , Phosphorylation , Protein Conformation , Protein Structure, Tertiary , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/ultrastructure , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/ultrastructure , Syk Kinase
6.
Biochim Biophys Acta ; 1774(12): 1493-9, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18021750

ABSTRACT

The cytoplasmic Syk kinase plays key roles in immune responses and comprises two N-terminal regulatory Src homology 2 (SH2) domains followed by a catalytic region. Atomic structures of these domains have only been solved in isolation. To gain insights into the three-dimensional structure of full-length Syk, we have used single-particle electron microscopy. Syk acquires a closed conformation resembling the inhibited structure of Zap-70, another member of the Syk family. Such configuration suggests an inhibition of the N-terminal domains on its catalytic activity. The phosphotyrosine binding pockets of both SH2 domains are not occluded and they could interact with other phosphoproteins.


Subject(s)
Imaging, Three-Dimensional , Intracellular Signaling Peptides and Proteins/chemistry , Microscopy, Electron/methods , Protein-Tyrosine Kinases/chemistry , Animals , Intracellular Signaling Peptides and Proteins/isolation & purification , Intracellular Signaling Peptides and Proteins/physiology , Models, Molecular , Protein Conformation , Protein-Tyrosine Kinases/isolation & purification , Protein-Tyrosine Kinases/physiology , Rats , Syk Kinase
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