scholarly journals Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Aimin Yang ◽  
Supansa Pantoom ◽  
Yao-Wen Wu
Keyword(s):  
Legionella Pneumophila ◽  
Lipid Binding ◽  
Effector Protein ◽  
Intracellular Pathogen ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Acyl Chains ◽  
Pathogenic Microbes ◽  
Lir Motif ◽  
Lipid Binding Site

Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.

Identification of the Lipid-Binding Site of Phosphatidylcholine-Transfer Protein with Phosphatidylcholine Analogs Containing Photactivable Carbene Precursors

1983 ◽  
Vol 132 (2) ◽  
pp. 441-449 ◽  
Author(s):  
Jan WESTERMAN ◽  
Karel W. A. WIRTZ ◽  
Theo BERKHOUT ◽  
Laurens L. M. DEENEN ◽  
Ramachandran RADHAKRISHNAN ◽  
...  
Keyword(s):  
Binding Site ◽  
Lipid Binding ◽  
Transfer Protein ◽  
Phosphatidylcholine Transfer Protein ◽  
Lipid Binding Site

scholarly journals Phospholipid transfer proteins revisited

1997 ◽  
Vol 324 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Karel. W. A WIRTZ
Keyword(s):  
Mammalian Cells ◽  
Lipid Transfer Protein ◽  
Fusion Reaction ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
Cell Functions ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) (identical with sterol carrier protein 2) belong to the large and diverse family of intracellular lipid-binding proteins. Although these two proteins may express a comparable phospholipid transfer activity in vitro, recent studies in yeast and mammalian cells have indicated that they serve completely different functions. PI-TP (identical with yeast SEC14p) plays an important role in vesicle flow both in the budding reaction from the trans-Golgi network and in the fusion reaction with the plasma membrane. In yeast, vesicle budding is linked to PI-TP regulating Golgi phosphatidylcholine (PC) biosynthesis with the apparent purpose of maintaining an optimal PI/PC ratio of the Golgi complex. In mammalian cells, vesicle flow appears to be dependent on PI-TP stimulating phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis. This latter process may also be linked to the ability of PI-TP to reconstitute the receptor-controlled PIP2-specific phospholipase C activity. The nsL-TP is a peroxisomal protein which, by its ability to bind fatty acyl-CoAs, is most likely involved in the β-oxidation of fatty acids in this organelle. This protein constitutes the N-terminus of the 58 kDa protein which is one of the peroxisomal 3-oxo-acyl-CoA thiolases. Further studies on these and other known phospholipid transfer proteins are bound to reveal new insights in their important role as mediators between lipid metabolism and cell functions.

scholarly journals High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2)

1999 ◽  
Vol 339 (1) ◽  
pp. 193-199 ◽  
Author(s):  
Tobias B. DANSEN ◽  
Jan WESTERMAN ◽  
Fred S. WOUTERS ◽  
Ronald J. A. WANDERS ◽  
Arie van HOEK ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid Transfer Protein ◽  
Fatty Acyl ◽  
Lipid Binding ◽  
Tryptophan Residue ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Specific Lipid ◽  
Long Chain ◽  
Lipid Binding Site

Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to β-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12–β-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the β-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible.

The roles of the human lipid-binding proteins ORP9S and ORP10S in vesicular transport

2005 ◽  
Vol 83 (5) ◽  
pp. 631-636 ◽  
Author(s):  
Gregory D Fairn ◽  
Christopher R McMaster
Keyword(s):  
Binding Protein ◽  
Vesicular Transport ◽  
Lipid Binding ◽  
Yeast Cells ◽  
Phospholipid Transfer Protein ◽  
Related Protein ◽  
Oxysterol Binding Protein ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Lipid Binding Proteins

Inactivation of the yeast oxysterol binding protein related protein (ORP) family member Kes1p allows yeast cells to survive in the absence of Sec14p, a phospholipid transfer protein required for cell viability because of the role it plays in transporting vesicles from the Golgi. We expressed human ORP9S and ORP10S in yeast lacking Sec14p and Kes1p function, and found that ORP9S completely complemented Kes1p function, whereas ORP10S possessed only a weak ability to replace Kes1p function. Purified ORP9S protein bound several phosphoinositides, whereas ORP10 bound specifically to phosphatidylinositol 3-phosphate. The combined evidence demonstrates that only a subset of human ORP proteins can function as negative regulators of Golgi-derived vesicular transport.Key words: phospholipid, Saccharomyces cerevisiae, Golgi, vesicular transport, oxysterol binding protein related protein.

Bioinformatics of the TULIP domain superfamily

2011 ◽  
Vol 39 (4) ◽  
pp. 1033-1038 ◽  
Author(s):  
Klaus O. Kopec ◽  
Vikram Alva ◽  
Andrei N. Lupas
Keyword(s):  
Binding Protein ◽  
Lipid Binding ◽  
Phospholipid Transfer Protein ◽  
Hypothetical Proteins ◽  
Hormone Binding ◽  
Lipopolysaccharide Binding Protein ◽  
Transfer Protein ◽  
Unicellular Eukaryotes ◽  
Phospholipid Transfer ◽  
Lipopolysaccharide Binding

Proteins of the BPI (bactericidal/permeability-increasing protein)-like family contain either one or two tandem copies of a fold that usually provides a tubular cavity for the binding of lipids. Bioinformatic analyses show that, in addition to its known members, which include BPI, LBP [LPS (lipopolysaccharide)-binding protein)], CETP (cholesteryl ester-transfer protein), PLTP (phospholipid-transfer protein) and PLUNC (palate, lung and nasal epithelium clone) protein, this family also includes other, more divergent groups containing hypothetical proteins from fungi, nematodes and deep-branching unicellular eukaryotes. More distantly, BPI-like proteins are related to a family of arthropod proteins that includes hormone-binding proteins (Takeout-like; previously described to adopt a BPI-like fold), allergens and several groups of uncharacterized proteins. At even greater evolutionary distance, BPI-like proteins are homologous with the SMP (synaptotagmin-like, mitochondrial and lipid-binding protein) domains, which are found in proteins associated with eukaryotic membrane processes. In particular, SMP domain-containing proteins of yeast form the ERMES [ER (endoplasmic reticulum)-mitochondria encounter structure], required for efficient phospholipid exchange between these organelles. This suggests that SMP domains themselves bind lipids and mediate their exchange between heterologous membranes. The most distant group of homologues we detected consists of uncharacterized animal proteins annotated as TM (transmembrane) 24. We propose to group these families together into one superfamily that we term as the TULIP (tubular lipid-binding) domain superfamily.

scholarly journals AMPylation Is Critical for Rab1 Localization to Vacuoles Containing Legionella pneumophila

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Camille A. Hardiman ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Host Protein ◽  
Effector Protein ◽  
Intracellular Pathogen ◽  
Effective Strategy ◽  
Nucleotide Exchange ◽  
Content Type ◽  
Cell Functions ◽  
Membrane Bound

ABSTRACTLegionella pneumophilais an intracellular pathogen that resides within a membrane-bound compartment that is derived from vesicles exiting the endoplasmic reticulum (ER). To create this compartment, these bacteria use a type IV secretion system to deliver effector proteins that subvert host cell functions. SeveralLegionellaeffector proteins modulate the function of the host protein Rab1, which is a GTPase that is recruited to theLegionella-containing vacuole (LCV). Here, we examined which of the Rab1-directed enzymatic activities displayed byLegionellaeffectors are important for localizing the Rab1 protein to the LCV membrane. The guanine nucleotide exchange factor (GEF) domain in the effector protein DrrA (SidM) was essential for Rab1 recruitment to the LCV and Rab1 AMPylation by the nucleotidyltransferase domain in DrrA was important for Rab1 retention.Legionellaorganisms producing mutant DrrA proteins that were severely attenuated for GEF activityin vitroretained the ability to localize Rab1 to the LCV. Rab1 localization to the LCV mediated by these GEF-defective mutants required AMPylation. Importantly, we found that efficient localization of Rab1 to the LCV occurred when Rab1 GEF activity and Rab1 AMPylation activity were provided by separate proteins. Rab1 phosphocholination (PCylation) by the effector protein AnkX, however, was unable to substitute for Rab1 AMPylation. Lastly, the defect in Rab1 localization to the LCV in AMPylation-deficient strains ofLegionellawas partially suppressed if the GTPase-activating protein (GAP) LepB was eliminated. Thus, our data indicate that AMPylation of Rab1 is an effective strategy to maintain this GTPase on the LCV membrane.IMPORTANCEActivities that enable the intracellular pathogenLegionella pneumophilato subvert the function of the host protein Rab1 were investigated. Our data show that a posttranslational modification called AMPylation is critical for maintaining a pool of Rab1 on the LCV membrane. AMPylation of Rab1 led to the accumulation of GTP-bound Rab1 on the LCV membrane by protecting the protein from inactivation by GAPs. Importantly, PCylation of Rab1 by theLegionellaeffector protein AnkX was neither necessary nor sufficient to maintain Rab1 on the LCV, indicating that AMPylation and PCylation represent functionally distinct activities. We conclude that modification of Rab1 by AMPylation is an effective strategy to spatially and temporally regulate the function of this GTPase on a membrane-bound organelle.

scholarly journals Legionella pneumophila Excludes Autophagy Adaptors from the Ubiquitin-Labeled Vacuole in Which It Resides

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Titilayo O. Omotade ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Effector Protein ◽  
Intracellular Pathogen ◽  
Intracellular Pathogens ◽  
Content Type ◽  
Acting Mechanism ◽  
Autophagy Pathway ◽  
Adaptor Recruitment ◽  
Dependent Mechanism

ABSTRACT Xenophagy targets intracellular pathogens for destruction by the host autophagy pathway. Ubiquitin chains are conjugated to xenophagic targets and recruit multiple autophagy adaptors. The intracellular pathogen Legionella pneumophila resides in a vacuole that is ubiquitinated; however, this pathogen avoids xenophagic detection. Here, the mechanisms by which L. pneumophila can prevent the host xenophagy pathway from targeting the vacuole in which it resides were examined. Ubiquitin-labeled vacuoles containing L. pneumophila failed to recruit autophagy adaptors by a process that was independent of RavZ function. Coinfection studies were conducted using a strain of Listeria monocytogenes that served as a robust xenophagic target. Legionella pneumophila infection blocked xenophagic targeting of L. monocytogenes by a RavZ-dependent mechanism. Importantly, when coinfection studies were conducted with a RavZ-deficient strain of L. pneumophila, L. monocytogenes was targeted by the host xenophagy system but vacuoles containing L. pneumophila avoided targeting. Enhanced adaptor recruitment to the vacuole was observed by using a strain of L. pneumophila in which all of the effector proteins in the SidE family were deleted; however, this strain was still not targeted by the host autophagy pathway. Thus, there are at least two pathways by which L. pneumophila can disrupt xenophagic targeting of the vacuole in which it resides. One mechanism involves global disruption of the host autophagy machinery by the effector protein RavZ. A second cis-acting mechanism prevents the binding of autophagy adaptors to the ubiquitin-decorated surface of the L. pneumophila-containing vacuole.

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