The trans-sialidase from the african trypanosome Trypanosoma brucei.

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.     

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

First structural glimpse at a bacterial Ser/Thr protein phosphatase

In this issue of Structure, we describe the crystal structure of the Ser/Thr protein phosphatase PstP from Mycobacterium tuberculosis, opening new perspectives to understand the putative roles of "eukaryotic-like" signaling elements in bacteria.     

Substrate-induced Conformational Changes in the Essential Peripheral Membrane-associated Mannosyltransferase PimA from Mycobacteria: IMPLICATIONS FOR CATALYSIS*

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the β-phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic α-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.Glycans are not only one of the major components of the cell but also are essential molecules that modulate a variety of important biological processes in all living organisms. Glycans are used primarily as energy storage and metabolic intermediates as well as being main structural constituents in bacteria and plants. Moreover, as a consequence of protein and lipid glycosylation, glycans generate a significant amount of structural diversity in biological systems. This structural information is particularly apparent in molecular recognition events including cell-cell interactions during critical steps of development, the immune response, host-pathogen interactions, and tumor cell metastasis. Most of the enzymes encoded in eukaryotic/prokaryotic/archaeans genomes that are responsible for the biosynthesis and modification of glycan structures are GTs³ (1). Here we have focused in the phosphatidyl-myo-inositol mannosyltransferase A (PimA), an essential enzyme of mycobacterial growth that initiates the biosynthetic pathway of key structural elements and virulence factors of Mycobacterium tuberculosis, the phosphatidyl-myo-inositol mannosides (PIM) lipomannan and lipoarabinomannan (2–5). This amphitropic enzyme catalyzes the transfer of a Manp residue from GDP-Man to the 2-position of PI to form phosphatidyl-myo-inositol monomannoside (PIM₁) on the cytoplasmic side of the plasma membrane (2) (Fig. 1).Open in a separate windowFIGURE 1.PIM₁ biosynthesis in mycobacteria. PimA transfers a Manp residue from GDP-Man to the 2-position of the myo-inositol ring of PI to form PIM₁ (where DAG is di-acyl-glycerol, and INS-P is 1-l-myo-inositol phosphate). The reaction occurs with retention of the anomeric configuration of the sugar donor.Although considerable progress has been made in recent years in understanding the mode of action of GTs at the molecular level, the mechanisms that govern recognition of lipid acceptors and membrane association of peripheral membrane-binding GTs remains poorly understood. GTs can be classified as either “inverting” or “retaining” enzymes according to the anomeric configuration of the reaction substrates and products. A single displacement mechanism in which a general base assists in the activation of the acceptor substrate for nucleophilic attack by the sugar donor is well established for inverting enzymes (6, 7). In contrast, the catalytic mechanism for retaining enzymes, including PimA, remains unclear. By analogy with glycosylhydrolases, a double displacement mechanism via the formation of a covalent glycosyl-enzyme intermediate was first proposed (8). However, in the absence of direct evidence of a viable covalent intermediate, an alternative mechanism known as the S_Ni “internal return” has been suggested where phospho-sugar donor bond breakage and sugar-acceptor bond formation occur in a concerted, but necessarily stepwise manner on the same face of the sugar (6, 9). Only two protein topologies have been found for nucleotide-diphospho-sugar-dependent enzymes among the first 30 GT sequence-based families (10) (see the carbohydrate-active enzymes (CAZy) data base) for which three-dimensional structures have been reported (11). These topologies are variations of “Rossmann-like” domains and have been defined as GT-A (12) and GT-B (13). Both inverting and retaining enzymes were found in GT-A and GT-B folds, indicating that there is no correlation between the overall fold of GTs and their catalytic mechanism. The primary sequence of PimA contains the GPGTF (glycogen phosphorylase/GT) motif, a signature present in enzymes of the GT-B fold (14). GT-B proteins do not use divalent cations and consist of two Rossmann-like (β-α-β) domains separated by a deep fissure. Therefore, an important interdomain movement has been predicted in some members of this superfamily during catalysis, including MurG (15), glycogen synthase (16),and the myo-inositol 1-phosphate N-acetylglucosaminyltransferase MshA (17).To perform their biochemical functions, membrane-binding GTs interact with membranes by two different mechanisms. Whereas integral membrane GTs are permanently attached through transmembrane regions (e.g. hydrophobic α-helices) (18) peripheral membrane-binding GTs temporarily bind membranes by (i) a stretch of hydrophobic residues exposed to bulk solvent, (ii) electropositive surface patches that interact with acidic phospholipids (e.g. amphipathic α-helices), and/or (iii) protein-protein interactions (19–22). A close interaction of the enzyme with membranes might be a strict requirement for PI modification by PimA. We recently solved the crystal structure of PimA from Mycobacterium smegmatis (MsPimA) in complex with the donor substrate GDP-Man (23, 24). The notion of a membrane-associated protein via electrostatic interactions is consistent with the finding of an amphipathic α-helix and surface-exposed hydrophobic residues in the N-terminal domain of MsPimA. Despite the fact that sugar transfer is catalyzed between the mannosyl group of the GDP-Man donor and the myo-inositol ring of PI, the enzyme displays an absolute requirement for both fatty acid chains of PI in order for the transfer reaction to take place. Furthermore, PimA was able to bind monodisperse PI, but its transferase activity was stimulated by high concentrations of nonsubstrate anionic surfactants, indicating that the reaction requires a lipid-water interface. We thus proposed a model of interfacial catalysis in which PimA recognizes the fully acylated substrate PI with its polar head within the catalytic cleft and the fatty acid moieties only partially sequestered from the bulk solvent (24).This study describes a detailed investigation of the lipid acceptor binding site and the conformational properties of PimA in solution. Using a combination of limited proteolysis, isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), analytical ultracentrifugation (AUC) and site-directed mutagenesis, we propose a plausible model for substrates recognition and binding. The implications of this model for the comprehension of the early steps of PIM biosynthesis and the catalytic mechanism of other members of the peripheral membrane-binding GT family are discussed.     

Allosteric activation of the protein kinase PDK1 with low molecular weight compounds

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate phosphoinositide-dependent protein kinase 1 (PDK1) by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.     

The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.     

CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2^LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.     

Adsorption of α-amylase and Starch on Porous Zinc Oxide Nanosheet: Biophysical Study

Food Biophysics - Engineered biocatalyst and its desired products using nanotechnology has intensified the research in food industries. Zinc oxide (ZnO) nanosheet is designed and prepared; the...