The NF-B family member RelB has many properties not shared by other family members such as restricted subunit association and lack of regulation by the classical IB proteins. We show that the protein level of RelB is significantly reduced in the absence of p100 and reduced even more when both p100 and p105 are absent. RelB stabilizes itself by directly interacting with p100, p105, and their processed products. However, RelB forms complexes with its partners using different interaction modes. Although the C-terminal ankyrin repeat domain of p105 is not involved in the RelB-p105 complex formation, all domains and flexible regions of each protein are engaged in the RelB-p100 complex. In several respects the RelB-p52 and RelB-p100 complexes are unique in the NF-B family. The N-terminal domain of p100/p52 interacts with RelB but not RelA. The transcriptional activation domain of RelB, but not RelA, directly interacts with the processing region of p100. These unique protein-protein contacts explain why RelB prefers p52 as its dimeric partner for transcriptional activity and is retained in the cytoplasm as an inhibited complex by p100. This association-mediated stabilization of RelB implies a possible role for RelB in the processing of p100 into p52.The dimeric NF-B transcription factors are formed from five family members, p50 (NF-B1), RelA (p65), p52 (NF-B2), c-Rel, and RelB. These proteins share an ϳ300-residue long homologous region located near the N terminus. This element, referred to as the Rel homology region (RHR), 5 is responsible for DNA binding, dimerization, inhibitor binding, and nuclear localization. p50 and p52 are the processed products of precursor proteins, p105 and p100, respectively (1, 2). RelA and c-Rel homo-and heterodimers are tightly regulated by a class of inhibitor proteins known as IB through the formation of stable IB-NF-B complexes that are unable to bind DNA. Activation of these dimers requires degradation of IB. A large number of stimuli activate IB degradation through phosphorylation of IB by IB kinase (IKK) leading to ubiquitination, 26 S proteasome recruitment, and degradation of IB by the proteasome (3, 4). Signaling pathways leading to NF-B activation through degradation of classical IB proteins (IB␣, IB␤, and IB⑀) are classified as the canonical pathways.RelB displays characteristics that are not shared by the other NF-B subunits: 1) Prototypical IB proteins do not regulate RelB-containing NF-B dimers (5, 6). 2) The RelB homodimer does not have DNA binding activity, suggesting that unlike other members, RelB may not form a stable detectable homodimer in vivo (7). 3) RelB has a N-terminal extension, known as the leucine zipper (LZ) domain because of the presence of a leucine-rich heptad repeat that is not present in other NF-B family members (8). 4) The x-ray crystal structure of the RelB dimerization domain (DD) revealed an intertwined domain swapped arrangement of the two monomers, suggesting that this RelB domain fold might be unstable and is stabilized by domain intertwining (9...

Inflammation of the temporomandibular joint (TMJ) can alter behavioral responses such as food intake and mobilize stress hormones. The hypothesis of this study was that food intake and diurnal corticosterone analysis can be used as indicators of adjuvant-induced TMJ inflammation. Groups of rats received adjuvant or no injections at the beginning of the resting (AM) or activity (PM) phase. Forty-eight hours (early) or 6 weeks (late) after adjuvant injection, plasma corticosterone was assayed and food intake was recorded. Food intake was suppressed up to 4 days post-injection. As expected, the non-injected group showed low AM and high PM corticosterone. AM corticosterone was elevated, but PM corticosterone was attenuated in both early- and late-stage-injected rats. A computerized pair-fed experiment showed that adjuvant-induced hypophagia did not alter corticosterone levels. Meal pattern analysis revealed decreased food intake due to a decrease in the number of meals taken. Notably, meal size remained the same but meal duration increased. This model demonstrated that food intake and stress hormone analysis could be used as indicators for sequelae of adjuvant-induced TMJ inflammation.

No abstract

Activation of a large multisubunit protein kinase, called the inhibitor kappaB kinase (IKK) complex, is central to the induction of the family of transcription factors nuclear factor kappaB. IKK is comprised of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory IKKgamma subunit. It is known that the catalytic IKKbeta and regulatory IKKgamma subunits associate through interactions mediated by the N-terminal region of IKKgamma and an 11-mer peptide located near the C-terminus of IKKbeta. In this study, we have defined the minimal IKKgamma segment that binds IKKbeta and determined the binding affinity of the IKKbeta/IKKgamma complex. We identified that the N-terminal segment spanning residues 40-130 of IKKgamma binds the IKKbeta C-terminal domain (residues 665-756) with Kd approximately 25 nM. Several smaller N-terminal IKKgamma deletion mutants within the N-terminal 130 residues, although in some cases retained IKKbeta binding activity, showed a tendency to aggregate and formed covalently linked complexes. However, expansion of the C-terminus of these fragments to residue 210 completely changed the solution behavior of the IKKgamma N-terminus without affecting the IKKbeta binding affinity. We also found that the IKKbeta C-terminal domain formed a dimer in solution and the basic unit of the IKKbeta/IKKgamma complex was a dimer/dimer.

Cannabinoids are antinociceptive in animal models of acute pain, tissue injury and nerve injury induced nociception and act via their cognate receptors, cannabinoid receptor 1 and 2. This review examines the underlying biology of the endocannabinoids and behavioural, neurophysiological, neuroanatomical evidence supporting the notion of pain modulation by these ligands with a focus on the current evidence encompassing the pharmacological characterization of CB1 agonists in this therapy. Separating the psychotropic effects of CB1 agonists from their therapeutic benefits is the major challenge facing researchers in the field today and with the discovery of peripherally acting agonists there seems to be a ray of hope emerging for the diverse potential therapeutic applications of this class of ligands.

The active site lysine residue, K256, involved in Schiff's base linkage with pyridoxal-5'-phosphate (PLP) in sheep liver recombinant serine hydroxymethyltransferase (rSHMT) was changed to glutamine or arginine by site-directed mutagenesis. The purified K256Q and K256R SHMTs had less than 0.1% of catalytic activity with serine and H4folate as substrates compared to rSHMT. The mutant enzymes also failed to exhibit the characteristic visible absorbance spectrum (lambda(max) 425 nm) and did not produce the quinonoid intermediate (lambda(max) 495 nm) upon the addition of glycine and H4folate. The mutant enzymes were unable to catalyze aldol cleavage of beta-phenylserine and transamination of D-alanine. These results suggested that the mutation of the lysine had resulted in the inability of the enzyme to bind to the cofactor. Therefore, the K256Q SHMT was isolated as a dimer and the K256R SHMT as a mixture of dimers and tetramers which were converted to dimers slowly. On the other hand, rSHMT was stable as a tetramer for several months, further confirming the role of PLP in maintenance of oligomeric structure. The mutant enzymes also failed to exhibit the increased thermal stability upon the addition of serine, normally observed with rSHMT. The enhanced thermal stability has been attributed to a change in conformation of the enzyme from open to closed form leading to reaction specificity. The mutant enzymes were unable to undergo this conformational change probably because of the absence of bound cofactor.

No abstract

Melanin concentrating hormone receptor 1 (MCHR1) antagonists have potential for the treatment of obesity and several CNS disorders. In the preceding article, we have described a novel series of quinazolines as MCHR1 antagonists and demonstrated in vivo proof of principle with an early lead. Herein we describe the detailed SAR and SPR studies to identify an optimized lead candidate having good efficacy in a sub-chronic DIO model with a good cardiovascular safety window.