Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.     

Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes

All primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1) vpr gene. HIV-1 vpr encodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2 arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains a vpr homologue, which encodes a 118-amino-acid protein. SIVagm vpr is structurally and functionally related to HIV-1 vpr. The present study focuses on how three specific functions (transactivation, induction of G2 arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagm vpr. While our study supports previous reports demonstrating a causal relationship between induction of G2 arrest and transactivation for HIV-1 vpr, we demonstrate that the same is not true for SIVagm vpr. Transactivation by SIVagm vpr is independent of cell cycle perturbation. In addition, we show that induction of G2 arrest is necessary for the induction of apoptosis by HIV-1 vpr but that the induction of apoptosis by SIVagm vpr is cell cycle independent. Finally, while SIVagm vpr retains its transactivation function in human cells, it is unable to induce G2 arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagm vpr are species specific. Taken together, our results suggest that while the multiple functions of vpr are conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.     

Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems: comparison with the recombinant enzyme purified from Escherichia coli.

Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for biochemical and physical characterization. However, the effective use of heterologous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" protein. For human sulfotransferases, little attention has been paid to this despite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine-metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharomyces cerevisiae, COS-7, and V79 cells, by comparison of Km values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expression. This is the first report of heterologous expression of a cytosolic sulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell types, and the Km for dopamine under the conditions applied was approximately 1 microM in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE, electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with Mr approximately 68,000 as determined by light scattering and sedimentation analysis. Mass spectrometry revealed two species with experimentally determined masses of 34,272 and 34,348 which correspond to the native protein with either one or two 2-mercaptoethanol adducts. We conclude that the enzyme expressed in prokaryotic and eukaryotic heterologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations.     

Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance

This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.     

Ultrastructure of the mature spermatozoa of caecilians (Amphibia: Gymnophiona)

The spermatozoa of Gymnophiona show the following autapomorphies: 1) penetration of the distal centriole by the axial fiber; 2) presence of an acrosomal baseplate; 3) presence of an acrosome seat (flattened apical end of nucleus); and 4) absence of juxta-axonemal fibers. The wide separation of the plasma membrane bounding the undulating membrane is here also considered to be apomorphic. Three plesiomorphic spermatozoal characters are recognized that are not seen in other Amphibia but occur in basal amniotes: 1) presence of mitochondria with a delicate array of concentric cristae (concentric cristae of salamander spermatozoa differ in lacking the delicate array); 2) presence of peripheral dense fibers associated with the triplets of the distal centriole; and 3) presence of a simple annulus (a highly modified, elongate annulus is present in salamander sperm). The presence of an endonuclear canal containing a perforatorium is a plesiomorphic feature of caecilian spermatozoa that is shared with urodeles, some basal anurans, sarcopterygian fish, and some amniotes. Spermatozoal synapomorphies are identified for 1) the Uraeotyphlidae and Ichthyophiidae, and 2) the Caeciliidae and Typhlonectidae, suggesting that the members of each pair of families are more closely related to each other than to other caecilians. Although caecilian spermatozoa exhibit the clear amphibian synapomorphy of the unilateral location of the undulating membrane and its axial fiber, they have no apomorphic characters that suggest a closer relationship to either the Urodela or Anura.     

Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatment-induced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulse-chase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.     

The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase

The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus. CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft. From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146. Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar. It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance. Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins. It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft.