Shedding of endogenous Interleukin-6 receptor (IL-6R) is governed by ADAM proteases while a full-length IL-6R isoform localizes to circulating microvesicles [Immunology]

September 10th, 2015 by

Generation of the soluble Interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine Interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings - or even in vivo - remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.
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Structural insights into cargo recognition by the yeast PTS1-receptor [Protein Structure and Folding]

September 10th, 2015 by

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 μM. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high-resolution mass spectrometry. Most cross-links were found with TPR6, TPR7 as well as the 7C-loop of Pex5p. Surface plasmon resonance (SPR) analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD of the interaction of truncated Pcs60p and Pex5p was in a range of 7.7 μM. ITC and SPR measurements revealed monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept: firstly, cargo recognition and initial tethering by the PTS1-receptor Pex5p and secondly lock-in of receptor and cargo.

Eisosomes regulate PI(4,5)P2 cortical clusters and MAP kinase signaling upon osmotic stress [Signal Transduction]

September 10th, 2015 by Kabeche, R., Madrid, M., Cansado, J., Moseley, J. B.

Eisosomes are multi-protein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P2 regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast S. pombe. Activation of the MAPK Pmk1 was impaired by mutations in the PI(5)-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P2 clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI(5)-kinase Its3, and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P2 clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.

The Q-Soluble-N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-Associated Membrane Proteins (VAMPs) [Cell Biology]

September 10th, 2015 by

SNAREs constitute the core machinery of intracellular membrane fusion but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with TGN VAMP4 and post-Golgi VAMPs 7 and 8. SNAP-47 also interacted with ER and Golgi Syntaxin 5 and with Syntaxin 1 in the absence of Munc18a, when Syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway.
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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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