Exposure of myeloid dendritic cells to exogenous or endogenous IL-10 during maturation determines their longevity

Dendritic cells (DC) are essential for the initiation of primary adaptive immune responses, and their functionality is strongly down-modulated by IL-10. Both innate and adaptive immune signals trigger the up-regulation of antiapoptotic Bcl-2 family members to facilitate the survival of DCs after maturation. However, whether IL-10 alters the expression of apoptotic-related genes in maturing DCs has not been determined. In this study, we demonstrate that spontaneous apoptosis rapidly occurred in myeloid DCs exposed to exogenous IL-10 upon maturation. Microarray analysis indicates that IL-10 suppressed the induction of three antiapoptotic genes, bcl-2, bcl-x, and bfl-1, which was coincident with the increased sensitivity of mature DCs to spontaneous apoptosis. IL-10 markedly inhibited the accumulation of steady state Bcl-2 message and protein in myeloid DCs activated through TLRs or TNFR family members, whereas exogenous IL-10 affected Bcl-x(L) expression in a moderate manner. In contrast, bcl-2 expression of plasmacytoid DCs was less sensitive to the effects of IL-10. We further show that autocrine IL-10 significantly limited the longevity of myeloid DCs and altered the expression kinetics of Bcl-2 but not Bcl-x(L) in maturing DCs. We conclude that the degree of IL-10 exposure and/or the level of endogenous IL-10 production upon myeloid DC maturation play a critical role in determining DC longevity. This regulatory mechanism of IL-10 is associated with the dynamic control of antiapoptotic Bcl-2 proteins.     

Specific protein domains mediate cooperative assembly of HuR oligomers on AU-rich mRNA-destabilizing sequences

The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.     

The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein

The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.     

Two distinct modes of processive kinesin movement in mixtures of ATP and AMP-PNP

An enzyme is frequently conceived of as having a single functional mechanism. This is particularly true for motor enzymes, where the necessity for tight coupling of mechanical and chemical cycles imposes rigid constraints on the reaction pathway. In mixtures of substrate (ATP) and an inhibitor (adenosine 5'-(beta,gamma-imido)triphosphate or AMP-PNP), single kinesin molecules move on microtubules in two distinct types of multiple-turnover "runs" that differ in their susceptibility to inhibition. Longer (less susceptible) runs are consistent with movement driven by the alternating-sites mechanism previously proposed for uninhibited kinesin. In contrast, kinesin molecules in shorter runs step with AMP-PNP continuously bound to one of the two active sites of the enzyme. Thus, in this mixture of substrate and inhibitor, kinesin can function as a motor enzyme using either of two distinct mechanisms. In one of these, the enzyme can accomplish high-duty-ratio processive movement without alternating-sites ATP hydrolysis.     

Dynamics and function in a bacterial ABC transporter: simulation studies of the BtuCDF system and its components

ABC transporters are integral membrane proteins which couple the energy of ATP hydrolysis to the translocation of solutes across cell membranes. BtuCD is a approximately 1100-residue protein found in the inner membrane of Gram-negative bacteria which transports vitamin B12. Vitamin B12 is bound in the periplasm by BtuF, which delivers the solute to the periplasmic entrance of the transporter protein complex BtuCD. Molecular dynamics simulations of the BtuCD and BtuCDF complexes (in a lipid bilayer) and of the isolated BtuD and BtuF proteins (in water) have been used to explore the conformational dynamics of this complex transport system. Overall, seven simulations have been performed, with and without bound ATP, corresponding to a total simulation time of 0.1 micros. Binding of ATP drives closure of the nucleotide-binding domains (NBDs) in BtuD in a symmetrical fashion, but not in BtuCD. It seems that ATP constrains the flexibility of the NBDs in BtuCD such that their closure may only occur upon binding of BtuF to the complex. Upon introduction of BtuF, and concomitant with NBD association, one ATP-binding site displays a closure, while the opposite site remains relatively unchanged. This asymmetry may reflect an initial step in the "alternating hydrolysis" mechanism and is consistent with measurements of nucleotide-binding stoichiometries. Principal components analysis of the simulation of BtuCD reveals motions that are comparable to those suggested in current transport models.     

Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia

This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O(2), respectively) or normoxia (21% O(2)) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2(-/-) mice compared with MMP-2(+/+) mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2(+/+) mice but not in MMP-2(-/-) mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.     

Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3

A series of quinazolinone-derived inhibitors of the CXCR3 receptor have been synthesized and their affinity for the receptor evaluated. Compounds were evaluated in a (125)I-IP10 displacement assay and in in vitro cell migration assays to IP10, ITAC, and MIG using human peripheral blood mononuclear cells.     

Finding NEMO: nestedness engendered by mutualistic organization in anemonefish and their hosts

The interaction structure of mutualistic relationships, in terms of relative specialization of the partners, is important to understanding their ecology and evolution. Analyses of the mutualistic interaction between anemonefish and their host sea anemones show that the relationship is highly nested in structure, generalist species interacting with one another and specialist species interacting mainly with generalists. This supports the hypothesis that the configuration of mutualistic interactions will tend towards nestedness. In this case, the structure of the interaction is at a much larger scale than previously hypothesized, across more than 180 degrees of longitude and some 60 degrees of latitude, probably owing to the pelagic dispersal capabilities of these species in a marine environment. Additionally, we found weak support for the hypothesis that geographically widespread species should be more generalized in their interactions than species with small ranges. This study extends understanding of the structure of mutualistic relationships into previously unexplored taxonomic and physical realms, and suggests how nestedness analysis can be applied to the conservation of obligate species interactions.