CTLA4 Autoimmunity-Associated Genotype Contributes to Severe Pulmonary Tuberculosis in an African Population

The gene of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA4), a negative regulator of T lymphocytes, contains a single-nucleotide polymorphism (SNP) at position +6230A->G (ct60A->G), which has been found associated with several autoimmune diseases and appears to reduce T-cell inhibitory activity. In Ghana, West Africa, we compared the frequencies of CTLA4 +6230 A/G and 6 haplotype-tagging SNPs in 2010 smear-positive, HIV-negative patients with pulmonary tuberculosis (TB) and 2346 controls matched for age, gender and ethnicity. We found no difference in allele frequencies between cases and controls. However, +6230A and a distinct CTLA4 haplotype and a diplotype comprising the +6230A allele were significantly less frequent among cases with large opacities in chest radiographs compared to those with small ones (P_{corrected [cor]} = 0.002, P_cor = 0.00045, P = 0.0005, respectively). This finding suggests that an increased T-cell activity associated with the CTLA4 +6230G allele contributes to pathology rather than to protection in pulmonary TB.     

The ubiquitin C‐terminal hydrolase UCH‐L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C‐terminal hydrolase UCH‐L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH‐L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH‐L1‐negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH‐L1 controls the early membrane‐associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c‐cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2‐ and AKT‐dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH‐L1_C90S. These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH‐L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria‐based drug delivery systems.     

The effect of substrate modification on binding of porcine pancreatic alpha amylase: hydrolysis of modified amylose containing D-allose residues

A modified amylose containing 10% of tritiated D-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified D-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that D-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of D-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of D-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.     

Protoporphyrin Treatment Modulates Susceptibility to Experimental Autoimmune Encephalomyelitis in miR-155-Deficient Mice

We previously identified heme oxygenase 1 (HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155 ^-/- CD4⁺ T cells to promote antigen-driven inflammation after adoptive transfer in antigen-expressing recipients. Protoporphyrins are molecules recognized for their modulatory effect on HO-1 expression and function. In the present study, we investigated the effect of protoporphyrin treatment on the development of autoimmunity in miR-155-deficient mice. MiR-155-mediated control of HO-1 expression in promoting T cell-driven chronic autoimmunity was confirmed since HO-1 inhibition restored susceptibility to experimental autoimmune encephalomyelitis (EAE) in miR-155-deficient mice. The increased severity of the disease was accompanied by an enhanced T cell infiltration into the brain. Taken together, these results underline the importance of miR-155-mediated control of HO-1 expression in regulating the function of chronically-stimulated T cells in EAE.     

Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.     

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.     

5 alpha-androstane-3 beta,17 beta-diol hydroxylating enzymes in stroma and epithelium of human benign prostatic hyperplasia (BPH)

As enzymatic hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) may be a factor in controlling the 5 alpha-dihydrotestosterone (DHT) content in the prostate, we were interested in activity and distribution of these enzymes in epithelium and stroma of human benign prostatic hyperplasia (BPH). The enzyme activities were measured after mechanical separation of BPH tissue from 15 patients of various ages into stroma and epithelium, and optimization of the in vitro transformation of 3 beta-diol to hydroxylated products, which were analyzed by HPLC. The main results were: (1) 3 beta-diol was hydroxylated at C-7 alpha, C-7 beta, C-6 alpha, and C-6 beta. (2) The mean Michaelis constant Km (nM +/- SEM) for hydroxylation at C-7 alpha(beta) (168 +/- 21) was significantly lower than at C-6 alpha(beta) (601 +/- 43) without differences between stroma and epithelium. (3) Hydroxylation at alpha position dominated significantly over that at beta. (4) The mean maximal metabolic rate Vmax (pmol . mg protein-1 . h-1) of hydroxylation at C-6 alpha was about 7-fold lower in stroma (3.4 +/- 0.2) than in epithelium (23.8 +/- 4.1), concerning the other hydroxylations, Vmax was about 1.6-fold lower in stroma. (5) With increasing age of the patients there was a significant decrease of the 3 beta-diol hydroxylation in stroma and epithelium. It is discussed that the significantly lower activity of 3 beta-diol hydroxylation in stroma compared to epithelium and the decrease of activity with increasing age might potentiate the DHT accumulation in stroma of BPH.     

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors.

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.     

Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli.

A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with Tn1000 and Tn5 IS50L::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hly, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxy-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (Mr 504) to maltoheptaose (Mr 1,152) and was totally abolished by dextran 4 (Mr 4,000). This result and the observed influx of [14C]sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin.