A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis

Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 A resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.     

The folded modules of aggrecan G3 domain exert two separable functions in glycosaminoglycan modification and product secretion.

Aggrecan is the major proteoglycan in the extracellular matrix of cartilage. A notable exception is nanomelic cartilage, which lacks aggrecan in its matrix. The example of nanomelia and other evidence leads us to believe that the G3 domain plays an important role in aggrecan processing, and it has indeed been confirmed that G3 allows glycosaminoglycan (GAG) chain attachment and product secretion. However, it is not clear how G3, which contains at least a carbohydrate recognition domain (CRD) and a complement binding protein (CBP) motif, plays these two functional roles. The present study was designed to dissect the mechanisms of this phenomenon and specially 1) to determine the effects of various cysteine residues in GAG modification and product secretion as well as 2) to investigate which of the two processing events is the critical step in the product processing. Our studies demonstrated that removal of the two amino-terminal cysteines in the CRD motif and the single cysteine in the amino terminus of CBP inhibited secretion of CRD and CBP. Use of the double mutant CRD construct also allowed us to observe a deviation from the usual strict coupling of GAG modification and product secretion steps. The presence of a small chondroitin sulfate fragment overcame the secretion-inhibitory effects once the small chondroitin sulfate fragment was modified by GAG.     

Homocysteine alterations in experimental cholestasis and its subsequent cirrhosis

Homocysteine (Hcy), an intermediate in methionine metabolism, has been proposed to be involved in hepatic fibrogenesis. Impaired liver function can alter Hcy metabolism. The aim of the present study was to determine plasma Hcy alterations in acute obstructive cholestasis and the subsequent biliary cirrhosis. Cholestasis was induced by bile duct ligation and sham-operated and unoperated rats were used as controls. The animals were studied on the days 7th, 14th, 21st and 28th after the operation. Plasma Hcy, cysteine, methionine, nitric oxide (NO) and liver S-adenosyl-methionine (SAM), S-adenosyl-homocysteine (SAH), SAM to SAH ratio and glutathione were measured. Chronic L-NAME treatment was also included in the study. Plasma Hcy concentrations were transiently elevated by the day 14th after bile duct ligation (P < 0.01) and subsequently returned to control levels. Similar relative fluctuations in plasma Hcy were observed in BDL rats after intraperitoneal methionine overload. Plasma methionine, cysteine and nitrite and nitrate were significantly increased after bile duct ligation. SAM to SAH ratio was diminished by the 1st week of cholestasis and remained significantly decreased throughout the study. These events were accompanied by a decrease in GSH to GSSG ratio in the liver. Chronic L-NAME treatment improved SAM to SAH ratio and prevented the elevation of plasma Hcy and methionine (P < 0.05) while couldn't influence the other parameters. In conclusion, this study demonstrates alterations in plasma Hcy and liver SAM and SAH contents in precirrhotic stages and in secondary biliary cirrhosis, for the first time. In addition, we observed that plasma Hcy concentrations in BDL rats follow a distinct pattern of alteration from what has been previously reported in other models of cirrhosis. NO overproduction may contribute to plasma Hcy elevation and liver SAM depletion after cholestasis.     

T cell-mediated graft-versus-leukemia reactions after allogeneic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation represents the only curative approach for many hematological malignancies. During the last years the impact of the conditioning regimen has been re-assessed. With the advent of reduced-intensity conditioning the paradigm has changed from cytoreduction executed by high-dose radio-chemotherapy to immunological surveillance of leukemia by donor cells. Distinct subsets of T cells and NK cells contribute to graft-versus-leukemia reactions. So far, cytotoxic T lymphocytes are the mainstay of allogeneic immunotherapy. Here, we summarise the current knowledge of T cell-mediated graft-versus-leukemia reactions and present results from pre-clinical and clinical studies of T cell-based adoptive immunotherapy. We address the issues of feasibility and specificity of adoptive immunotransfer from a clinical point of view and discuss the prerequisites for successful clinical applications. Finally, the prospects for immunological research that have evolved with the increasing use of reduced-intensity conditioning and allogeneic stem cell transplantation are highlighted.     

High performance liquid chromatographic determination of azithromycin in serum using fluorescence detection and its application in human pharmacokinetic studies

A fast and sensitive high-performance liquid chromatographic method for determination of azithromycin in human serum using fluorescence detection was developed. The drug and an internal standard (clarithromycin) were extracted from serum using n-hexan and subjected to pre-column derivatization with 9-fluorenylmethyl chloroformate as labeling agent. Analysis was performed on a phenyl packing material column with sodium phosphate buffer containing 2 ml/l triethylamine (pH 5.9) and methanol (29:71, v/v) as the mobile phase. The standard curve was linear over the range of 10-500 ng/ml of azithromycin in human serum. The means between-days precision were from 13.3% (for 10 ng/ml) to 2% (500 ng/ml) and the within-day precision from 11.9 to 1.7% determined on spiked samples. The accuracy of the method was 100.7-107.2% (between days) and 100.3-107.8% (within day). The limit of quantification was 10 ng/ml. This method was applied in a bioequivalence study of four different azithromycin preparations in 12 healthy volunteers.     

A structural inventory of native ribosomal ABCE1‐43S pre‐initiation complexes

In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub‐complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP‐binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre‐initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1‐containing pre‐initiation complexes by cryo‐EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide‐binding domains, while interacting with the N‐terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C‐terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near‐complete molecular picture of the architecture and sophisticated interaction network of the 43S‐bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.     

Dynamic probabilistic threshold networks to infer signaling pathways from time-course perturbation data

Background

Network inference deals with the reconstruction of molecular networks from experimental data. Given N molecular species, the challenge is to find the underlying network. Due to data limitations, this typically is an ill-posed problem, and requires the integration of prior biological knowledge or strong regularization. We here focus on the situation when time-resolved measurements of a system’s response after systematic perturbations are available.

Results

We present a novel method to infer signaling networks from time-course perturbation data. We utilize dynamic Bayesian networks with probabilistic Boolean threshold functions to describe protein activation. The model posterior distribution is analyzed using evolutionary MCMC sampling and subsequent clustering, resulting in probability distributions over alternative networks. We evaluate our method on simulated data, and study its performance with respect to data set size and levels of noise. We then use our method to study EGF-mediated signaling in the ERBB pathway.

Conclusions

Dynamic Probabilistic Threshold Networks is a new method to infer signaling networks from time-series perturbation data. It exploits the dynamic response of a system after external perturbation for network reconstruction. On simulated data, we show that the approach outperforms current state of the art methods. On the ERBB data, our approach recovers a significant fraction of the known interactions, and predicts novel mechanisms in the ERBB pathway.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-250) contains supplementary material, which is available to authorized users.     

Development of efficient miniprep transformation methods for Artemisia annua using Agrobacterium tumefaciens and Agrobacterium rhizogenes

Extensive studies have been carried out for the optimization of regeneration and transformation conditions for both Agrobacterium tumefaciens- and Agrobacterium rhizogenes-mediated transformation of the highly medicinal plant Artemisia annua. Most protocols describe laborious transformation procedures requiring no less than 3 mo to obtain transgenic plants. This study reports rapid and efficient protocols for A. tumefaciens- and A. rhizogenes-mediated transformation of A. annua, which were equally effective for transformation of Artemisia dubia. In both transformation procedures, stem explants responded best for maximal production of transformed plants and hairy roots. In the case of A. tumefaciens-mediated transformation, stem explants were pre-cultured for 2 d followed by infection with A. tumefaciens strain LBA4404 for 48 h. A. annua explants showed maximal transformation rate (43.5%) on half-strength Murashige and Skoog medium containing 40 mg/L kanamycin in only 20 d. The same method was tested using a related species A. dubia and resulted in a transformation rate of 41.3%, demonstrating that this protocol is efficient and genotype-independent. In the case of A. rhizogenes-mediated transformation for the production of hairy root cultures, in vitro-grown stem explants were infected with a single colony of A. rhizogenes strain LBA9402 by creating incisions at different places of the stem explants, which resulted in production of hairy roots in only 7 d. The method was tested in both A. annua and A. dubia, which resulted in transformation rates of 90 and 87.5%, respectively. Integration of the transgene and copy number was confirmed by PCR and Southern blot analyses, respectively. The miniprep transformation protocols developed for both A. tumefaciens- and A. rhizogenes-mediated transformation are simple, efficient, and potentially applicable to other species of Artemisia for transfer of pharmaceutically important genes.     

Time to Seroconversion in HIV-Exposed Subjects Carrying Protective versus Non Protective KIR3DS1/L1 and HLA-B Genotypes

Natural killer (NK) cells play a role in the clearance of viral infections. Combinations of alleles at the polymorphic HLA-B locus and the NK cell surface killer immunoglobulin-like receptor locus KIR3DL1/S1 have been shown to influence time to AIDS in HIV-infected individuals and risk of seroconversion in HIV exposed seronegative (HESN) subjects. Here, we assessed time to seroconversion or duration of seronegative status in a group of 168 HIV exposed individuals, including 74 seroconverters and 94 HESN based on carriage or not of KIR3DL1/S1/HLA-B genotypes previously shown to be associated with protection from infection and/or slow time to AIDS. KIR3DL1/S1 genotyping was performed by sequence-specific primer polymerase chain reaction using two pairs of specific primers for each locus. The MHC class IB locus was typed to four-position resolution to resolve Bw4 and Bw6 alleles and the amino acid present at position 80. KIR3DL1/S1 heterozygotes became HIV infected significantly faster than KIR3DS1 homozygotes. Individuals who carried both KIR3DS1 and Bw4*80I did not remain HIV seronegative longer than those from a control group who were homozygous for HLA-Bw6 and carried no HLA-A locus Bw4 alleles Subjects who were *h/*y+B*57 showed a trend towards slower time to serconversion than those with other KIR3DL1 homozygous and KIR3DL1/S1 heterozygous genotypes. Thus, KIR3DS1 homozygosity is associated with protection from HIV infection while co-carriage of KIR3DS1 and Bw4*80I is not. The requirements for protection from HIV infection can differ from those that influence time to AIDS in HIV infected individuals.