Analyses of Protein Turnover at the Cell Plate by Fluorescence Recovery After Photobleaching During Cytokinesis.

Membrane trafficking is central to cell plate construction during plant cytokinesis. Studies on cell plate formation can provide answers to basic biology questions including molecular mechanisms of membrane trafficking, tissue patterning, and cytoskeletal dynamics. Consequently, a detailed understanding of cytokinesis depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles and delivery of proteins to the developing and maturing plate. This chapter describes a pipeline based on fluorescence recovery after photobleaching (FRAP) to measure and analyze turnover of peripheral or transmembrane proteins on the cell plate. The approach described here can also be applied in other biological contexts.

ERα36-GPER1 Collaboration Inhibits TLR4/NFκB-Induced Pro-Inflammatory Activity in Breast Cancer Cells.

Inflammation is important for the initiation and progression of breast cancer. We have previously reported that in monocytes, estrogen regulates TLR4/NFκB-mediated inflammation via the interaction of the Erα isoform ERα36 with GPER1. We therefore investigated whether a similar mechanism is present in breast cancer epithelial cells, and the effect of ERα36 expression on the classic 66 kD ERα isoform (ERα66) functions. We report that estrogen inhibits LPS-induced NFκB activity and the expression of downstream molecules TNFα and IL-6. In the absence of ERα66, ERα36 and GPER1 are both indispensable for this effect. In the presence of ERα66, ERα36 or GPER1 knock-down partially inhibits NFκB-mediated inflammation. In both cases, ERα36 overexpression enhances the inhibitory effect of estrogen on inflammation. We also verify that ERα36 and GPER1 physically interact, especially after LPS treatment, and that GPER1 interacts directly with NFκB. When both ERα66 and ERα36 are expressed, the latter acts as an inhibitor of ERα66 via its binding to estrogen response elements. We also report that the activation of ERα36 leads to the inhibition of breast cancer cell proliferation. Our data support that ERα36 is an inhibitory estrogen receptor that, in collaboration with GPER1, inhibits NFκB-mediated inflammation and ERα66 actions in breast cancer cells.

Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1.

Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti-inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30/G-protein estrogen receptor 1, in a sex-independent manner. 17-ß-Estradiol inhibits the LPS-induced IL-6 inflammatory response, resulting in inhibition of NF-κB transcriptional activity. This is achieved via a direct physical interaction of ligand-activated estrogen receptor-α 36-kDa splice variant with the p65 component of NF-κB in the nucleus. G-protein coupled receptor 30/G-protein estrogen receptor 1, which also physically interacts with estrogen receptor-α 36-kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL-6 expression. However, its activation does not mimic the effect of estrogens, on neither IL-6 nor NF-κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage-dependent inflammatory processes.

LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis.

The genomes of Bacillus cereus and its closest relative Bacillus anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524). Only a few members of this family have been biochemically characterized including N-acetylglucosaminylphosphatidyl inositol (GlcNAc-PI), 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins), N,N'-diacetylchitobiose (GlcNAc(2)) and lipoglycopeptide antibiotic de-N-acetylases. All these enzymes share a common feature in that they de-N-acetylate the N-acetyl-D-glucosamine (GlcNAc) moiety of their substrates. The bc1534 gene has previously been cloned and expressed in Escherichia coli. The recombinant enzyme was purified and its 3D structure determined. In this study, the bc3461 gene from B. cereus ATCC14579 was cloned and expressed in E. coli. The recombinant enzymes BC1534 (EC 3.5.1.-) and BC3461 were biochemically characterized. The enzymes have different molecular masses, pH and temperature optima and broad substrate specificity, de-N-acetylating GlcNAc and N-acetylchito-oligomers (GlcNAc(2), GlcNAc(3) and GlcNAc(4)), as well as GlcNAc-1P, N-acetyl-D-glucosamine-1 phosphate; GlcNAc-6P, N-acetyl-D-glucosamine-6 phosphate; GalNAc, N-acetyl-D-galactosamine; ManNAc, N-acetyl-D-mannosamine; UDP-GlcNAc, uridine 5'-diphosphate N-acetyl-D-glucosamine. However, the enzymes were not active on radiolabeled glycol chitin, peptidoglycan from B. cereus, N-acetyl-D-glucosaminyl-(beta-1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) or N-acetyl-D-GlcN-Nalpha1-6-D-myo-inositol-1-HPO(4)-octadecyl (GlcNAc-I-P-C(18)). Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc(2) revealed that GlcNAc(2) is the favored substrate for both native enzymes. Based on the recently determined crystal structure of BC1534, a mutational analysis identified functional key residues, highlighting their importance for the catalytic mechanism and the substrate specificity of the enzyme. The catalytic efficiencies of BC1534 variants were significantly decreased compared to the native enzyme. An alignment-based tree places both de-N-acetylases in functional categories that are different from those of other LmbE proteins.

Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase.

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E.

Purification, crystallization and preliminary X-ray analysis of the peptidoglycan N-acetylglucosamine deacetylase BC1960 from Bacillus cereus in the presence of its substrate (GlcNAc)6.

The peptidoglycan N-acetylglucosamine (GlcNAc) deacetylase BC1960 from Bacillus cereus (EC 3.5.1.33), an enzyme consisting of 275 amino acids, was crystallized in the presence of its substrate (GlcNAc)(6). The crystals belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 92.7, c = 242.9 A and four molecules in the asymmetric unit. A complete data set was collected at 100 K to a resolution of 2.38 A using synchrotron radiation.

Crystal structure of the BcZBP, a zinc-binding protein from Bacillus cereus.

Bacillus cereus is an opportunistic pathogenic bacterium closely related to Bacillus anthracis, the causative agent of anthrax in mammals. A significant portion of the B. cereus chromosomal genes are common to B. anthracis, including genes which in B. anthracis code for putative virulence and surface proteins. B. cereus thus provides a convenient model organism for studying proteins potentially associated with the pathogenicity of the highly infectious B. anthracis. The zinc-binding protein of B. cereus, BcZBP, is encoded from the bc1534 gene which has three homologues to B. anthracis. The protein exhibits deacetylase activity with the N-acetyl moiety of the N-acetylglucosamine and the diacetylchitobiose and triacetylchitotriose. However, neither the specific substrate of the BcZBP nor the biochemical pathway have been conclusively identified. Here, we present the crystal structure of BcZBP at 1.8 A resolution. The N-terminal part of the 234 amino acid protein adopts a Rossmann fold whereas the C-terminal part consists of two beta-strands and two alpha-helices. In the crystal, the protein forms a compact hexamer, in agreement with solution data. A zinc binding site and a potential active site have been identified in each monomer. These sites have extensive similarities to those found in two known zinc-dependent hydrolases with deacetylase activity, MshB and LpxC, despite a low degree of amino acid sequence identity. The functional implications and a possible catalytic mechanism are discussed.

Purification, crystallization and preliminary characterization of a putative LmbE-like deacetylase from Bacillus cereus.

The Bacillus cereus BC1534 protein, a putative deacetylase from the LmbE family, has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Crystals of the 26 kDa protein grown from MPD and acetate buffer belong to space group R32, with unit-cell parameters a = b = 76.7, c = 410.5 A (in the hexagonal setting). A complete native data set was collected to a resolution of 2.5 A from a single cryoprotected crystal using synchrotron radiation. As BC1534 shows significant sequence homology with an LmbE-like protein of known structure from Thermus thermophilus, molecular replacement will be used for crystal structure determination.

Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis.

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(-) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(beta-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc(2-6) revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc(2-4)) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.