Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin, PilE [Protein Structure and Folding]

September 10th, 2015 by

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilEδ1-28 shows a typical type IV pilin fold, showing how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilXNm and PilVNm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilXNm and PilVNm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.

Phosphorylation of Serine 402 Regulates RacGAP Activity of FilGAP [Signal Transduction]

September 10th, 2015 by Morishita, Y., Tsutsumi, K., Ohta, Y.

FilGAP is a Rho GTPase-activating protein (GAP), which specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at S402 is localized to the cytoplasm but not at the cytoskeleton. Although S402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of S402 accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with Calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin whereas cells transfected with FilGAP S402A mutant was not affected by Calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP and phosphorylation of S402 may play a role in the regulation of cell spreading on fibronectin.

Saturation mutagenesis of the antithrombin reactive center loop P14 residue supports a 3-step mechanism of heparin allosteric activation involving intermediate and fully-activated states [Protein Structure and Folding]

September 10th, 2015 by

Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized fifteen antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild-type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations′ effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate and fully-activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully-activated state and the bulk of allosteric activation has occurred in the intermediate.
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Combinatorial and computational approaches to identify interactions of macrophage colony stimulating factor (M-CSF) and its receptor c-fms [Computational Biology]

September 10th, 2015 by

The molecular interactions between macrophage colony stimulating factor (M-CSF) and the tyrosine kinase receptor c-fms play a key role in the immune response, bone metabolism and the development of some cancers. Since no X-ray structure is available for the human M-CSF/c-fms complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-fms. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSFM) single mutants and screened this library to isolate variants with reduced affinity for c-fms using FACS. Sequencing yielded a number of single M-CSFM variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSFM structure and to classify the mutations into three groups: those that significantly decrease protein stability, those that destroy favorable intermolecular interactions, and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSFM and three variants were produced as soluble proteins, and their affinity and structure were analyzed; very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSFM residues critical for M-CSF/c-fms interactions, we have laid down the basis for a deeper understanding of the M-CSF/c-fms signaling mechanism and for the development of target specific therapeutic agents with the ability to sterically occlude the M-CSF/c-fms binding interface.
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Structure of a CGI-58 Motif Provides the Molecular Basis of Lipid Droplet Anchoring [Protein Structure and Folding]

September 8th, 2015 by

Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for beta-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain CGI-58`s ability to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N-terminus (residues 10-31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp21 and Trp25) and right (harboring Trp29) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp21 and Trp25 and two adjacent leucines. Trp29 serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm.

Thrombin Promotes Sustained Signaling and Inflammatory Gene Expression through the CDC25 and Ras Associating Domains of Phospholipase C-epsilon [Molecular Bases of Disease]

September 8th, 2015 by Dusaban, S. S., Kunkel, M. T., Smrcka, A. V., Brown, J. H.

Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein coupled receptor mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme's Ras associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses.
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Microsomal triglyceride transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin, but not glycosylceramide [Metabolism]

September 8th, 2015 by

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin, but normal hexosylceramide, lactosylceramide and different sphingosines compared with unaffected controls. Further, similar differential effects on plasma sphingolipids were seen in liver and intestine specific MTP knockout (L,I-Mttp-/-) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis, but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion, but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.
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Systematic analysis of mycobacterial acylation reveals first example of acylation-mediated regulation of enzyme activity of a bacterial phosphatase [Signal Transduction]

September 8th, 2015 by

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins is not studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys224. This residue is situated in a lid region that covers enzyme′s active site. Consequently, acetylation and succinylation negatively regulates the activity of PtpB.
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G0/G1 switch gene-2 regulates cardiac lipolysis [Metabolism]

September 8th, 2015 by

The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids (FAs) to prevent lipotoxicity, while TAG hydrolysis (lipolysis) remobilizes FAs from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G0/G1 switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild type and mutant mice we show that (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2-overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL-deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.

Na-H exchanger isoform-2 (NHE2) mediates butyrate-dependent Na+ absorption in dextran sulfate sodium (DSS)-induced colitis [Cell Biology]

September 8th, 2015 by Rajendran, V. M., Nanda Kumar, N. S., Tse, C. M., Binder, H. J.

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na+ absorption. Although colonic Na+ absorption is mediated by both epithelial Na+ channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na+ absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na+ absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na+ fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO3--dependent and butyrate-dependent Na+ absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO3--dependent Na+ absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO3--dependent and butyrate-dependent Na+ absorption, while DSS- induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO3--dependent) Na+ absorption. In in vivo loop studies HCO3--Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, while in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO3--Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-medated Na+ absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea.