Regulating Arp2/3-Dependent Actin Assembly by Collapsin Response Mediator Protein-1 [Cell Biology]

November 23rd, 2015 by Yu-Kemp, H.-C., Brieher, W. M.

Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3 dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3 dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own nor does it directly activate the Arp2/3 complex. Rather, CRMP- 1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation.

Runx1 Phosphorylation by Src Increases Trans-Activation via Augmented Stability, Reduced HDAC Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis [Signal Transduction]

November 23rd, 2015 by

Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification - increased stability, reduced HDAC interaction, and increased DNA-binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA-binding, in gel shift assays and as assessed by chromatin immunoprecipitation with the -14 kb Pu.1 or +37 kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.
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Lipid-Protein Interactions in the Regulated Betaine Symporter BetP Probed by Infrared Spectroscopy [Molecular Biophysics]

November 22nd, 2015 by Guler, G., Gartner, R. M., Ziegler, C., Mantele, W.

The Na+-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K+ during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behaviour of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidyl glycerol lipids to functional important parts of BetP suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K+ binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions especially with the C-terminal domain and the functional important gating helices TMH3 and TMH12 confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point towards a more general mechanism how active transport can be modified by dynamic lipid-protein interactions.

Optimal neutralization of Centruroides noxius venom is understood through a structural complex between two antibody fragments and the Cn2 toxin [Immunology]

November 20th, 2015 by

Nowadays, the trends in the research devoted to develop novel antidotes against scorpion stings have showed excellent results with the use of recombinant antibody fragments. The polyclonal character of commercial anti-venoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals we aimed to develop an equivalent recombinant anti-venom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpionCentruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously envenomed animals. The crystallographic structure of the ternary complex (scFv LR-Cn2-scFv RU1) allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.
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MicroRNA-424 Predicts a Role for {beta}-1,4 Branched Glycosylation in Cell Cycle Progression [Cell Biology]

November 20th, 2015 by Vaiana, C. A., Kurcon, T., Mahal, L. K.

MicroRNA (miRNA) regulation of protein expression plays an important role in mediating many cellular processes, from cell proliferation to cell death. The human microRNA hsa-miR-424 is upregulated in response to anti-proliferative cytokines, and directly represses cell cycle progression. Our lab recently established that miRNA can be used as a proxy to identify biological roles of glycosylation enzymes (glycogenes). Herein, we identify MGAT4A, OGT and GALNT13 as targets of miR-424. MGAT4A, but not the other two glycogenes, is regulated by miR-424 in the mammary epithelial cancer cell line MCF-7. Knockdown of MGAT4A in MCF-7 induces cell cycle arrest through decreasing CCND1 and CDC25A levels. Loss of MGAT4A does not affect levels of β-(1,6) branched N-glycans, arguing that this effect is not due to gross changes in N-glycan branching but rather is specific to the β-(1,4) branch. This is the first example of miR-424 regulation of glycosylation related genes, lending insight into one possible mechanism by which cells use specific branched glycosylation patterns to regulate cell cycle progression.

Increasing the Receptor Tyrosine Kinase EphB2 Prevents Amyloid-{beta}-induced Depletion of Cell-Surface Glutamate Receptors by a Mechanism that Requires EphB2’s PDZ-binding Motif and Neuronal Activity [Neurobiology]

November 20th, 2015 by Miyamoto, T., Kim, D., Knox, J. A., Johnson, E., Mucke, L.

Diverse lines of evidence suggest that amyloid-β peptides (Aβ) causally contribute to the pathogenesis of Alzheimer′s disease (AD), the most frequent neurodegenerative disorder. However, the mechanisms by which Aβ impairs neuronal functions remain to be fully elucidated. Previous studies showed that soluble Aβ oligomers interfere with synaptic functions by depleting NMDA-type glutamate receptors (NMDARs) from the neuronal surface and that overexpression of the receptor tyrosine kinase EphB2 can counteract this process. Through pharmacological treatments and biochemical analyses of primary neuronal cultures expressing wildtype or mutant forms of EphB2, we demonstrate that this protective effect of EphB2 depends on its PDZ-binding motif and the presence of neuronal activity, but not on its kinase activity. We further present evidence that EphB2′s protective effect may be mediated by the AMPA-type glutamate receptor (AMPAR) subunit GluA2, which can become associated with EphB2′s PDZ-binding motif through PDZ domain containing proteins and can promote the retention of NMDARs in the membrane. In addition, we show that the Aβ-induced depletion of surface NMDARs does not depend on several factors that have been implicated in the pathogenesis of Aβ-induced neuronal dysfunction, including aberrant neuronal activity, tau, prion protein (PrPC), and EphB2 itself. Thus, although EphB2 does not appear to be directly involved in the Aβ-induced depletion of NMDARs, increasing its expression may counteract this pathogenic process through a neuronal activity- and PDZ-dependent regulation of AMPARs.
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Cancer Cell Invasion in 3D collagen is Differentially Regulated by G{alpha}13 and Discoidin Domain Receptor 1-Par3 Signaling [Glycobiology and Extracellular Matrices]

November 20th, 2015 by

Cancer cells can invade in 3D collagen as single cells or as a cohesive group of cells that requires coordination of cell-cell junctions and the actin cytoskeleton. To examine the role of Gα13, a G12 family heterotrimeric G protein, in regulating cellular invasion in 3D collagen, we established a novel method to track cell invasion by membrane type 1-matrix metalloproteinase (MT1-MMP)-expressing cancer cells. We show that knockdown of Gα13 decreased MT1-MMP-driven proteolytic invasion in 3D collagen and enhanced E-cadherin-mediated cell-cell adhesion. E-cadherin knockdown reversed Gα13 siRNA-induced cell-cell adhesion, but failed to reverse the effect of Gα13 siRNA on proteolytic invasion. Instead, concurrent knockdown of E-cadherin and Gα13 led to an increased number of single cells rather than groups of cells. Significantly, knockdown of discoidin domain receptor 1 (DDR1), a collagen-binding protein that also co-localizes to cell-cell junctions, reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in 3D collagen. Knockdown of the polarity protein Par3, which can function downstream of DDR1, also reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in 3D collagen. Overall, we show that Gα13 and DDR1-Par3 differentially regulate cell-cell junctions and the actin cytoskeleton to mediate invasion in 3D collagen.
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The Structure of a T3SS Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing [Protein Structure and Folding]

November 20th, 2015 by

The Type 3 Secretion System (T3SS) and the bacterial flagellum are related pathogenicity associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface, to interact with the host cell and/or promote motility. A proposed "ruler" protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a "ball-and-chain" architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain does not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the Nterminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly the ruler protein's N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins show that this mechanism is likely conserved across systems.

Lipoprotein, LprI, of Mycobacterium tuberculosis acts as a lysozyme inhibitor [Bioenergetics]

November 20th, 2015 by

Mycobacterium tuberculosis (Mtb) executes numerous defence strategies for the successful establishment of infection under diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface localized lipoprotein, LprI, which is exclusively present in Mtb complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin, HbN) and both are synchronously up-regulated in Mtb during macrophage infection. Recombinant LprI, expressed in E. coli, exhibited strong binding (Kd ≤ 2 nM) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein labelled protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis Micrococcus lysodeikticus from lysozyme mediated hydrolysis. Expression of the lprI gene in M. smegmatis (8-10 folds) and survival during intracellular infection of peritoneal and monocyte derived macrophages, known to secrete lysozyme, and also in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2 to 3 folds in secondary cell lines in the presence of lysozyme in comparison to isogenic cells, expressing these genes individually. Thus, specific advantage against macrophage generated lysozyme, conferred by the combination of LprI-HbN during invasion of Mtb, may have vital implications on pathogenesis of tuberculosis.

Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity [Molecular Bases of Disease]

November 20th, 2015 by

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phospho-inositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in-vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical trichomonas isolates and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galecitin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.