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Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-α, whereas LT targets IL-10 and TNF-α production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-γ secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.

The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription factor activator protein 1 (AP1). This mutation simultaneously reduces polyomavirus transcription and DNA replication. Reversion of this mutation simultaneously restores binding of PEA1 and both DNA replication and transcription. Binding of a second protein, PEA2, adjacent to the PEA1 site at nt 5147-5155 is enhanced by PEA1 binding, suggesting that these proteins interact. A third protein, PEA3, binds to the sequence AGGAAG (nt 5133-5138) adjacent to the PEA1 binding site; integrity of this late-proximal PEA3 binding site or an additional earlyproximal site (nt 5228-5233) is important for enhancer function. We correlate binding of PEA1 and PEA2 with the induction of a DNase 1-hypersensitive site in polyomavirus minichromosomes isolated from mouse fibroblasts.The polyomavirus enhancer activates the viral early promoter in vivo and is required for viral DNA replication (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). It is composed of multiple functionally redundant DNA elements whose activities vary with cell type and growth state (2, 5-7, 9, 11, 12). These elements serve as binding sites for cellular proteins (13-20) that most likely help form initiation complexes at cis-linked origins and promoters (5,14,21).Two cellular proteins (15) bind to the polyomavirus enhancer at nucleotides (nt) 5139-5155 of the A3 strain (22) or nt 5114-5130 of the A2 strain (23). Polyomavirus enhancer A binding protein 1 (PEA1) binds to nt 5139-5145 (A2 strain nt 5114-5120), which make up the consensus sequence for the HeLa cell transcription factor activator protein 1 (AP1) (TGACTAA). AP1 activates the human metallothionein and simian virus 40 enhancers (24); it is most likely encoded by the human protooncogene c-jun (25), and it shares substantial sequence homology with the DNA-binding domains of yeast . PEA1 is probably the murine homolog of AP1 (21, 30).PEA2 binds to nt 5147-5154 (A2 strain nt 5122-5129), adjacent to the PEA1 binding site. An additional factor, polyomavirus enhancer B binding protein 1 (PEB1), binds to other enhancer sequences between nt 5180 and 5220 (A2 strain nt 5155-5195) (13,14,21), and several proteins- Because of the functional redundancy of the polyomavirus enhancer, we chose to inactivate multiple important elements by introducing numerous random point mutations. Using this approach, we identified several polyomaviruses whose DNA replication and transcription were greatly reduced because of point mutations in the enhancer (5). In this report we provide evidence for the involvement of PEA1 and PEA2, with a third factor, PEA3, ...

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C 1 and C 2 ). The first cytoplasmic domain of type I enzyme (IC 1 ) and the second cytoplasmic domain of type II enzyme (IIC 2 -⌬3, a construct in which 36 N-terminal amino acids of the C 2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in G s␣ -and forskolin-activated enzyme activity. The turnover number for G s␣ -and forskolin-stimulated enzyme activity of the complex between IC 1 and IIC 2 -⌬3 was 8.2 s ؊1 . The concentration of IIC 2 -⌬3 to achieve half-maximal activation of IC 1 was 0.8 and 1.3 M when stimulated by forskolin and G s␣ , respectively. The concentration of IIC 2 -⌬3 needed to complex with IC 1 was reduced 10-fold (0.08 M) when the enzyme was activated by both forskolin and G s␣ , suggesting that G s␣ and forskolin increased the affinity of the two cytoplasmic domains for each other.The enzymatic activity of adenylyl cyclase is the key step in regulating the intracellular cAMP concentration upon stimulation of a variety of hormones, neurotransmitters, and other regulatory molecules. There are at least nine distinct mammalian adenylyl cyclases which have a similar structure (Fig. 1A) (1-11). This includes two intensely hydrophobic domains (M 1 and M 2 ) and two ϳ40-kDa cytoplasmic domains (C 1 and C 2 ). The C 1 and C 2 domains contain sequences (C 1a and C 2a ) that are similar to each other and to other adenylyl and guanylyl cyclases (12, 13). Each isoform of adenylyl cyclase has its own distinct tissue distribution and unique regulatory properties, providing modes for different cells to respond diversely to similar stimuli (12,14).Membrane-bound adenylyl cyclases are expressed in small quantities, and the enzyme is labile and difficult to manipulate in detergent-containing solutions. To facilitate biochemical and structural analysis, a soluble adenylyl cyclase has been constructed by linking the C 1a and C 2a domains of type I and type II adenylyl cyclases, respectively (15). The resulting protein is sensitive to activation by G s␣ 1 and forskolin and to inhibition by P-site inhibitors, indicating the essential roles of C 1a and C 2a domains for catalysis and regulation. In this paper, we describe the expression and purification of the C 1a and C 2a domains of type I and type II adenylyl cyclase, respectively. Alone, each has no adenylyl cyclase activity; however, mixing of the two domains in vitro results in G s␣ -and forskolin-activated enzyme activity. EXPERIMENTAL PROCEDURESPlasmids-For construction of the expression plasmid vector pProEx-HAH6, the NcoI and EcoRI 4.9-kb fragment of pProEx-1 (Life Technologies, Inc.) was ligated with the phosphorylated linkers (5Ј-CATGCATCACCATCACCATCACGCGGCCGCCTACCCGTATGATGT-CCCGGATTACGCCGGAATTCCCATGGC and 5Ј-AATTGCCATGGGA-ATTCCGGCGTAATCCGGGACATCATACGGGTAGGCGGCCGCGTG-TGGTGATGGTGATG). Proper insertion of cDNA at the NcoI site of pProEx-HAH6 vector would result in the expres...

CC chemokine ligand 5 (CCL5) and CCL3 are critical for immune surveillance and inflammation. Consequently, they are linked to the pathogenesis of many inflammatory conditions and are therapeutic targets. Oligomerization and glycosaminoglycan (GAG) binding of CCL5 and CCL3 are vital for the functions of these chemokines. Our structural and biophysical analyses of human CCL5 reveal that CCL5 oligomerization is a polymerization process in which CCL5 forms rod-shaped, double-helical oligomers. This CCL5 structure explains mutational data and offers a unified mechanism for CCL3, CCL4, and CCL5 assembly into high-molecular-weight, polydisperse oligomers. A conserved, positively charged BBXB motif is key for the binding of CC chemokines to GAG. However, this motif is partially buried when CCL3, CCL4, and CCL5 are oligomerized; thus, the mechanism by which GAG binds these chemokine oligomers has been elusive. Our structures of GAG-bound CCL5 and CCL3 oligomers reveal that these chemokine oligomers have distinct GAG-binding mechanisms. The CCL5 oligomer uses another positively charged and fully exposed motif, KKWVR, in GAG binding. However, residues from two partially buried BBXB motifs along with other residues combine to form a GAG-binding groove in the CCL3 oligomer. The N termini of CC chemokines are shown to be involved in receptor binding and oligomerization. We also report an alternative CCL3 oligomer structure that reveals how conformational changes in CCL3 N termini profoundly alter its surface properties and dimer-dimer interactions to affect GAG binding and oligomerization. Such complexity in oligomerization and GAG binding enables intricate, physiologically relevant regulation of CC chemokine functions.signal transduction | CC chemokine | protein oligomerization | glycosaminoglycan | X-ray crystallography T he CC chemokines are a 28-member family of 8-to 14-kDa small-molecular-weight (MW) chemotactic cytokines with crucial roles in inflammation and infection (1, 2). Chemokine oligomerization and their interaction with glycosaminoglycans (GAGs), polysaccharides that are either free or attached to proteoglycans mostly on cell surface or in the extracellular matrix, is a coupled process that play key roles in chemokine functions (3-8). These include, but are not limited to, protection from proteolysis, regulation of chemotactic/haptatactic gradients to guide cell migration, transcytosis of chemokines across cells, and presentation to surface receptors of target cells, particularly under flow conditions. Most CC chemokines readily dimerize by themselves and form higher-MW complexes in the presence of GAGs (4). CC chemokine ligand 3 (CCL3) (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) are CCR5 ligands that are involved in diverse proinflammatory responses and are targeted for therapeutic innovations for human diseases including cancer, cardiovascular diseases, and HIV infection (9-12). Unlike other CC chemokines, these chemokines reversibly self-assemble into high-MW oligomers, up to >600 kDa in size (13,14). The pre...

Several forms of adenylyl cyclase (types I, II, V, and VI) have been expressed using the recombinant baculovirus expression system in Sf9 cells. The activation of type I adenylyl cyclase by forskolin and Gs alpha was not greater than additive. In contrast, there was synergistic activation of type II, V, and VI adenylyl cyclases by Gs alpha and forskolin. Gs alpha potentiated the effect of forskolin on type II adenylyl cyclase to the greatest extent. Type I and II adenylyl cyclases were photolabeled specifically by an iodinated photoaffinity derivative of forskolin ([125I]-6-AIPP-Fsk). Type I adenylyl cyclase was photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha only slightly increased the labeling efficiency. In contrast, type II adenylyl cyclase was not photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha greatly enhanced the labeling efficiency. Photolabeling of type V and VI adenylyl cyclases was detected only in the presence of Gs alpha. Neither calcium/calmodulin nor G protein beta gamma subunits modulated the photolabeling of type I or II adenylyl cyclases. Another iodinated derivative of forskolin, [125I]-6-IHPP-fsk, bound to Sf9 cell membranes expressing type I adenylyl cyclase with high affinity in a filtration binding assay, and the specific binding was not enhanced by the addition of Gs alpha. In contrast, specific binding of [125I]-6-IHPP-Fsk to membranes expressing type II adenylyl cyclase was detected only in the presence of Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Despite the demonstration that chronic morphine increases phosphorylation of multiple substrate proteins, their identity has, for the most part, remained elusive. Thus far, chronic morphine has not been shown to increase the phosphorylation of any identified effector protein. This is the first demonstration that persistent activation of opioid receptors has profound effects on phosphorylation of adenylyl cyclase (AC). A dramatic increase in phosphorylation of AC (type II family) was observed in ileum longitudinal muscle myenteric plexus preparations obtained from chronic morphine-treated guinea pigs. Analogous results were obtained when AC was immunoprecipitated using two differentially directed AC antibodies. The magnitude of the augmented AC phosphorylation was substantially attenuated by chelerythrine, a protein kinase C-selective inhibitor. These results suggest the potential relevance of increased phosphorylation (protein kinase C-mediated) of AC to opioid tolerant/dependent mechanisms. Because phosphorylation of AC isoforms (type II family) can significantly increase their stimulatory responsiveness to Gsalpha and Gbetagamma, this mechanism could underlie, in part, the predominance of opioid AC stimulatory signaling observed in opioid tolerant/dependent tissue. Moreover, in light of the fact that many G protein-coupled receptors signal through common effector proteins, this effect provides a mechanism for divergent consequences of chronic morphine treatment and could explain the well documented complexity of changes that accompany the opioid tolerant/dependent state.

Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.