Imma

Abstract: The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-PhrI sensory system activate ICEBs1 gene expression, excision and transfer by inactivating the ICEBs1 repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBs1-encoded anti-repressor ImmA (YdcM). ImmA was needed for the degradation of ImmR in B. subtilis. Coexpression of ImmA and ImmR in Escherichia coli or co-incubation of purified ImmA and ImmR resulted in site-specific cleavage of ImmR. Homologues of immR and immA are found in many mobile genetic elements. We found that the ImmA homologue encoded by B. subtilis phage phi105 is required for inactivation of the phi105 repressor (an ImmR homologue). ImmA-dependent proteolysis of ImmR repressors may be a conserved mechanism for regulating horizontal gene transfer.

Abstract: The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in Bacillus subtilis. The RecA-dependent SOS response and the RapI-PhrI cell sensory system activate ICEBs1 gene expression by stimulating cleavage of ImmR, the ICEBs1 immunity repressor, by the protease ImmA. We found that increasing the amount of wild-type ImmA in vivo caused partial derepression of ICEBs1 gene expression. However, during RapI-mediated derepression of ICEBs1 gene expression, ImmA levels did not detectably increase, indicating that RapI likely activates the protease ImmA by increasing its specific activity. We also isolated and characterized mutations in immA (immA(h)) that cause partial derepression of ICEBs1 gene expression in the absence of inducing signals. We obtained two types of immA(h) mutations: one type caused increased amounts of the mutant proteins in vivo but no detectable effect on specific activity in vitro; the other type had no detectable effect on the amount of the mutant protein in vivo but caused increased specific activity of the protein (as measured in vitro). Together, these findings indicate that derepression of ICEBs1 gene expression is likely caused by an increase in the specific activity of ImmA. Homologs of ImmA and ImmR are found in many mobile genetic elements, so the mechanisms that regulate ImmA-mediated cleavage of ImmR may be widely conserved.

Abstract: Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.

Abstract: Enhancement of endocannabinoid signaling has emerged as an attractive strategy for the treatment of pain. In addition to the well-characterized hydrolytic pathways, cyclooxygenase-2 (COX-2) mediated oxygenation is thought to be an alternative route for endocannabinoid metabolism and therefore provides a new avenue for drug intervention. In this study, we examined the therapeutic effect of indomethacin morpholinamide (IMMA), a novel substrate-selective COX-2 inhibitor, in the chronic constriction injury (CCI) mouse model. Treatment with IMMA significantly alleviated hyperalgesia and mechanical allodynia demonstrated by increased thermal withdrawal latency in Hargreaves test and tactile thresholds in Von Frey test. Accumulation of astrocytes and microglia in spinal cord dorsal horn and infiltration of macrophages into the dorsal root ganglion and sciatic nerve were reduced by drug treatment. Co-administration of the CB2 receptor antagonist, but not the CB1 receptor antagonist partially reversed the inhibitory effect of IMMA on pain sensitivity and inflammatory infiltrates. IMMA downregulated the mRNA expression of TNF-α and IL-1β and the production of IL-6 and MCP-1 proteins in the ipsilateral sciatic nerve. The enhanced NF-κB DNA binding activity in the CCI mouse dorsal spinal cord was also significantly reduced, suggesting that inactivation of NF-κB contributes to the anti-inflammatory property of IMMA. However, different from the previous reports showing that IMMA can increase endocannabinoids without interfering with arachidonic acid metabolism, treatment with IMMA failed to elevate the endogenous levels of AEA and 2-AG, but significantly reduced the production of prostaglandin E2 (PGE2). Furthermore, the mRNA expression of enzymes involved in PGE2 production, COX-2 and prostaglandin E synthase 2 in the ipsilateral sciatic nerve was also suppressed by IMMA treatment. Taken together, these results suggested that IMMA might exert anti-nociceptive effects through multiple mechanisms which include, but are not limited to, CB2 receptor activation and reduced PGE2 production.