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.

Arabidopsis Rab Geranylgeranyltransferases Demonstrate Redundancy and Broad Substrate Specificity in vitro [Plant Biology]

November 20th, 2015 by Shi, W., Zeng, Q., Kunkel, B. N., Running, M. P.

Posttranslational lipid modifications mediate the membrane attachment of Rab GTPases, facilitating their function in regulating intracellular vesicular trafficking. In Arabidopsis, most Rab GTPases have two C-terminal cysteines and potentially can be double geranylgeranylated by heterodimeric Rab geranylgeranyltransferases (Rab-GGTs). Genes encoding two putative α subunits and two putative β subunits of Rab-GGTs have been annotated in the Arabidopsis thaliana genome, but little is known about Rab-GGT activity in Arabidopsis. In this study, we demonstrate that four different heterodimers can be formed between putative Arabidopsis Rab-GGT α subunits RGTA1/RGTA2 and β subunits RGTB1/RGTB2, but only RGTA1-RGTB1 and RGTA1-RGTB2 exhibit bona fide Rab-GGT activity, and they are biochemically redundant in vitro. We hypothesize that RGTA2 function might be disrupted by a 12-amino acid insertion in a conserved motif. We present evidence that Arabidopsis Rab-GGTs may have preference for prenylation of C-terminal cysteines in particular positions. We also demonstrate that Arabidopsis Rab-GGTs can prenylate not only a great variety of Rab GTPases in the presence of Rab escort protein (REP), but, unlike Rab-GGT in yeast and mammals, can also certain non-Rab GTPases independent of REP. Our findings may help explain some of the phenotypes of Arabidopsis protein prenyltransferase mutants.

Advanced glycation end products (AGE) potently induce autophagy through activation of RAF kinase and NF-KAPPA B [Molecular Bases of Disease]

November 19th, 2015 by Verma, N., Manna, S. K.

Advanced glycation end products (AGE) accumulate in diabetic patients and aging people due to high amounts of 3- or 4-carbon sugars derived from glucose and thereby causing multiple consequences including inflammation, apoptosis, obesity and age-related disorders. It is important to understand the mechanism of AGE-mediated signaling leading to activation of autophagy (self-eating) that might result in obesity. We have detected AGE as one of the potent inducers of autophagy compared to doxorubicin and TNF. AGE-mediated autophagy is inhibited by suppression of PI3 kinase and potentiated by autophagosome maturation blocker, bafilomycin. It increases autophagy in different cell types and that correlates with the expression of it receptor, RAGE. LC3B, the marker for autophagosome is shown to increase upon AGE stimulation. AGE-mediated autophagy is suppressed partially by inhibitor of NF-κB, PKC, or ERK alone and significantly in combination. AGE increases SREBP activity that leads to increase in lipogenesis. Though AGE-mediated lipogenesis is affected by autophagy inhibitor, AGE-mediated autophagy is not influenced by lipogenesis inhibitor, suggesting that the turnover of lipid droplets overcomes the autophagic clearance. For the first time, we are providing data that AGE induces several cell signaling cascades, like NF-κB, PKC, ERK, and MAPK, which are involved in autophagy and simultaneously help in accumulating lipid droplets which are not effectively cleared by autophagy, thus follows obesity.

The Relay-Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II [Cell Biology]

November 19th, 2015 by Bloemink, M. J., Melkani, G. C., Bernstein, S. I., Geeves, M. A.

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1 we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase towards wild-type values. The S1- R759E construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke and involving a change in conformation at the relay/converter domain interface. Significantly the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modelling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP-binding and hydrolysis.