The importance of a critical protonation state and the fate of the catalytic steps in class A beta-lactamases and penicillin-binding proteins

Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.     

Regional isolation in the Balkan region: an analysis of craniofacial variation

Biological variation is investigated among contemporary Croatians, Bosnians, American whites, and other multitemporal Balkan populations (World War II Croatians, Macedonians, and Greeks) via multivariate statistics and distance measures of the craniofacial complex. This study demonstrates that there is considerable variation among groups of European ancestry. Bosnians and Croatians who are thought to be relatively homogenous and historically to originate from the same Slav ancestry show local variations. While environmental plasticity has been used to explain cranial changes among human groups, it does not adequately explain the variation observed between Bosnians and Croatians. It is an oversimplification to exclusively attribute the vast range of variability observed among local as well as geographic populations to environmental adaptations.     

Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery

The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed.     

INCA, a novel human caspase recruitment domain protein that inhibits interleukin-1beta generation

Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.     

Developmental regulation of peach ACC oxidase promoter--GUS fusions in transgenic tomato fruits

A genomic DNA sequence (PpACO1) encoding 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from peach (Prunus persica L. Batsch cv. Loring) was isolated. It has four exons interrupted by three introns and 2.9 kb of flanking region 5' of the translational start codon. Previous work with the cDNA demonstrated that accumulation of the peach ACO message correlated with increasing amounts of ethylene synthesized by the fruit as they ripened. To identify regulatory elements in the peach ACC oxidase gene, chimeric fusions between 403, 610, 901, 1319, 2141, and 2919 bp of the 5' flanking region of the PpACO1 sequence and the beta-glucuronidase (GUS) coding sequence were constructed and used to transform tomato (Lycopersicon esculentum [Mill] cv. Pixie). Fruits from the various promoter lines were analysed for GUS expression by histochemical GUS staining, GUS quantitative enzyme activity determination, and measuring the relative amounts of GUS mRNA. Constructs with the smallest promoter of 403 bp had significant GUS expression in fruit, but not in other tissues, indicating the presence of a region that affects tissue-specific expression. An increase in GUS expression was observed with promoters longer than 901 bp, indicating an enhancer region between -1319 and -901. The full-length promoter of 2919 bp directed GUS expression in the green stage of fruit development, and increased GUS expression as fruit matured, indicating a regulatory region between -2919 and -2141 that controls the temporal expression of the gene in fruit. Only the full-length promoter sequence demonstrated responsiveness to ethylene.     

Nonmyeloablative bone marrow transplantation of BXSB lupus mice using fully matched allogeneic donor cells from green fluorescent protein transgenic mice

Male BXSB mice, a mouse model of systemic lupus erythematosus, were given bone marrow transplants (BMT) at 20 wk of age using MHC-matched donor cells and nonmyeloablative conditioning (550 cGy irradiation). Transplanted mice and irradiation controls were followed for a period of 20 wk. Mice transgenic for green fluorescent protein were used as donors to allow tracking of donor cells and a determination of chimerism. Radiation controls had reduced renal pathology at 10 wk posttransplant, but not at 20 wk compared with untreated mice, while nonmyeloablative BMT mice had significantly reduced pathology at both time intervals. The monocytosis characteristic of older BXSB mice was also reduced by BMT, but the treatment did not prevent production of Ab to dsDNA. A stable chimerism of 24-40% donor CD45-positive cells was achieved in spleen and bone marrow, and there was no evidence of clinical graft vs host disease. Donor cells were detected in most recipient organs, notably the thymus and renal glomeruli. The results suggest that complete depletion of mature lymphocytes or of progenitor stem cells is not required to control lupus nephritis in BXSB mice.     

Transgenic expression of insulin receptor substrate 2 in murine B cells alters the cell density-dependence of IgE production in vitro and enhances IgE production in vivo

Previous studies have shown that insulin receptor substrate (IRS)1 and IRS2 mediate proliferative and antiapoptotic signaling through the IL-4R in 32D cells; however their role in regulating normal B cell responses is not clear. To investigate the role of IRS2 in normal B cell function, we developed IRS2 transgenic (Tg) mice on the C57BL/6 background. Western blot analysis revealed a 2-fold elevation in IRS2 protein levels in Tg(+) mice compared with littermate controls and a 3-fold increase in basal tyrosine phosphorylated IRS2 in the absence of IL-4 stimulation. IL-4-induced tyrosine phosphorylation of IRS2 was elevated in Tg(+) B cells, whereas IL-4-induced phosphorylation of STAT6 was similar between Tg(+) and Tg(-) B cells. Tg expression of IRS2 had little effect on IL-4-mediated proliferation and no effect on protection from apoptosis. However, production of IgE and IgG1 by Tg(+) B cells using standard in vitro conditions was diminished 50-60%. Because Ig production in vitro is known to be highly cell concentration-dependent, we performed experiments at different cell concentrations. Interestingly, at very low B cell concentrations (1000-5000 B cells/well), IgE and IgG1 production by Tg(+) B cells was greater than that of controls, whereas at higher cell concentrations (10,000-20,000 cells/well) Ig production by Tg(+) B cells was less than controls. Furthermore, in vivo immunization with OVA-alum or goat anti-IgD resulted in elevated serum IgE levels in the Tg(+) mice. These results indicate that overexpression of IRS2 alters the B cell intrinsic density-dependence of IgE and IgG1 production in vitro and enhances IgE responses in vivo.     

Terminal deoxynucleotidyltransferase deficiency decreases autoimmune disease in diabetes-prone nonobese diabetic mice and lupus-prone MRL-Fas(lpr) mice

The wide diversity of the T and B Ag receptor repertoires becomes even more extensive postneonatally due to the activity of TdT, which adds nontemplated N nucleotides to Ig and TCR coding ends during V(D)J recombination. In addition, complementarity-determining region 3 sequences formed in the absence of TdT are more uniform due to the use of short sequence homologies between the V, D, and J genes. Thus, the action of TdT produces an adult repertoire that is both different from, and much larger than, the repertoire of the neonate. We have generated TdT-deficient nonobese diabetic (NOD) and MRL-Fas(lpr) mice, and observed a decrease in the incidence of autoimmune disease, including absence of diabetes and decreased pancreatic infiltration in NOD TdT(-/-) mice, and reduced glomerulonephritis and increased life span in MRL-Fas(lpr) TdT(-/-) mice. Using tetramer staining, TdT(-/-) and TdT(+/+) NOD mice showed similar frequencies of the diabetogenic BDC 2.5 CD4(+) T cells. We found no increase in CD4(+)CD25(+) regulatory T cells in NOD TdT(-/-) mice. Thus, TdT deficiency ameliorates the severity of disease in both lupus and diabetes, two very disparate autoimmune diseases that affect different organs, with damage conducted by different effector cell types. The neonatal repertoire appears to be deficient in autoreactive T and/or B cells with high enough affinities to induce end-stage disease. We suggest that the paucity of autoreactive specificities created in the N region-lacking repertoire, and the resultant protection afforded to the newborn, may be the reason that TdT expression is delayed in ontogeny.     

Complex role of the IL-4 receptor alpha in a murine model of airway inflammation: expression of the IL-4 receptor alpha on nonlymphoid cells of bone marrow origin contributes to severity of inflammation

Recent studies have suggested the IL-4Ralpha expressed on lung epithelium is necessary for TH2-mediated goblet cell differentiation and mucus hypersecretion in a murine model of allergic lung disease. However, the IL-4Ralpha is expressed on numerous cell types that could contribute to the overall pathology and severity of asthma. The relative role of the receptor on these cells has not yet been conclusively delineated. To dissect the contribution of IL-4Ralpha in the development of pulmonary allergic responses, we generated murine radiation bone marrow (BM) chimeras. BM from IL-4Ralpha(+) or IL-4Ralpha(-) mice was transferred into recipient mice that expressed or lacked IL-4Ralpha. In the absence of IL-4Ralpha in recipient mice, there was no goblet cell metaplasia or mucus hypersecretion in response to OVA, even in the presence of TH2 cells and substantial eosinophilic infiltration. More importantly, we found that expression of the IL-4Ralpha on a nonlymphoid, MHC class II(+), BM-derived cell type contributes to the severity of inflammation and mucus production. These results suggest that IL-4 and IL-13 contribute to the development of allergic inflammation by stimulating a complex interaction between IL-4Ralpha(+) cell types of both bone marrow and non-bone marrow origin.