Loss of recognition by cross-reactive T cells and its relation to a C-terminus-induced conformational reorientation of an HLA-B*2705-bound peptide

The human major histocompatibility complex class I antigen HLA‐B*2705 binds several sequence‐related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross‐reactivity of cytotoxic T cells (CTL) against these HLA‐B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging‐in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross‐reactivity of these CTL with a peptide derived from voltage‐dependent calcium channel α1 subunit (pCAC, SRRWRRWNR), in which the C‐terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA‐B*2705:pCAC complex by X‐ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence‐related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F‐pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA‐B*2705 heavy chain near the N‐terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg‐Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR‐directed, HLA‐B*2705‐restricted CTL to cross‐react with HLA‐B*2705:pCAC complexes.     

The cell cycle gene MoCDC15 regulates hyphal growth, asexual development and plant infection in the rice blast pathogen Magnaporthe oryzae

Rice blast, caused by the pathogen Magnaporthe oryzae, is a serious hindrance to rice production and has emerged as an important model for the characterization of molecular mechanisms relevant to pathogenic development in plants. Similar to other pathogenic fungi, conidiation plays a central role in initiation of M.oryzae infection and spread over a large area. However, relatively little is known regarding the molecular mechanisms that underlie conidiation in M. oryzae. To better characterize these mechanisms, we identified a conidiation-defective mutant, ATMT0225B6 (MoCDC15(T-DNA)), in which a T-DNA insertion disrupted a gene that encodes a homolog of fission yeast cdc15, and generated a second strain containing a disruption in the same allele (ΔMoCDC15(T-DNA)). The cdc15 gene has been shown to act as a coordinator of the cell cycle in yeast. Functional analysis of the MoCDC15(T-DNA) and ΔMoCDC15(T-DNA) mutants revealed that MoCDC15 is required for conidiation, preinfection development and pathogenicity in M. oryzae. Conidia from these mutants were viable, but failed to adhere to hydrophobic surface, a crucial step required for subsequent pathogenic development. All phenotypic defects observed in mutants were rescued in a strain complemented with wild type MoCDC15. Together, these data indicate that MoCDC15 functions as a coordinator of several biological processes important for pathogenic development in M. oryzae.     

Involvement of intracellular Ca2+ in blue light-dependent proton pumping in guard cell protoplasts from Vicia faba

Recent studies have suggested that Ca²⁺/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells. As the increase in cytosolic concentration of Ca²⁺ is required for the activation of CaM and CaM-like proteins, the origin of the Ca²⁺ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba . BL-dependent proton pumping was affected neither by Ca²⁺ channel blockers nor by changes of Ca²⁺ concentration in the medium used for the GCPs. Addition of Ca²⁺ ionophores and an agonist to GCPs did not induce proton pumping. However, BL-dependent proton pumping was inhibited by 10 m M caffeine, which releases Ca²⁺ from the intracellular stores, and by 10 μ M 2,5-di-( tert -butyl)-1,4-benzohydroquinone (BHQ) and 10 μ M cyclopiazonic acid (CPA), inhibitors of Ca²⁺-ATPase in the sarcoplasmic and endoplasmic reticulum (ER). By contrast, the inhibitions were not observed by 10 μ M thapsigargin, an inhibitor of animal ER-type Ca²⁺-ATPase. The inhibitions by caffeine and BHQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA. From these results, we conclude that the Ca²⁺ thought to be required for BL-dependent proton pumping may originate from intracellular Ca²⁺ stores, most likely from ER in guard cells, and that this origin of Ca²⁺ may generate a stimulus-specific Ca²⁺ signal for stomatal opening.     

Transfer of exogenous cholesterol to microsomes of hepatocytes investigated with [3H]desmosterol tracer.

The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r. spectroscopy of molybdenum(V) in reduced forms of the enzyme. The resting enzyme gives no signals attributable to Mo(V). However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced. On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized. Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as 'signal A' and 'signal B', analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase. Signal A has gav. 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av. 1.3mT, whereas signal B shows no more than weak coupling to protons. Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl-. This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson [Biochem. J. (1983) 211, 227-236] that Cl- is a ligand of molybdenum in the species giving signal A.     

Impact of host cell line choice on glycan profile

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.     

Thematic Review Series: Recent Advances in the Treatment of Lysosomal Storage Diseases: Gene therapy for the neurological manifestations in lysosomal storage disorders

Over the past several years, considerable progress has been made in the development of gene therapy as a therapeutic strategy for a variety of inherited metabolic diseases, including neuropathic lysosomal storage disorders (LSDs). The premise of gene therapy for this group of diseases is borne of findings that genetic modification of a subset of cells can provide a more global benefit by virtue of the ability of the secreted lysosomal enzymes to effect cross-correction of adjacent and distal cells. Preclinical studies in small and large animal models of these disorders support the application of either a direct in vivo approach using recombinant adeno-associated viral vectors or an ex vivo strategy using lentiviral vector-modified hematopoietic stem cells to correct the neurological component of these diseases. Early clinical studies utilizing both approaches have begun or are in late-stage planning for a small number of neuropathic LSDs. Although initial indications from these studies are encouraging, it is evident that second-generation vectors that exhibit a greater safety profile and transduction activity may be required before this optimism can be fully realized. Here, I review recent progress and the remaining challenges to treat the neurological aspects of various LSDs using this therapeutic paradigm.     

Constant pH molecular dynamics of proteins in explicit solvent with proton tautomerism

pH is a ubiquitous regulator of biological activity, including protein‐folding, protein‐protein interactions, and enzymatic activity. Existing constant pH molecular dynamics (CPHMD) models that were developed to address questions related to the pH‐dependent properties of proteins are largely based on implicit solvent models. However, implicit solvent models are known to underestimate the desolvation energy of buried charged residues, increasing the error associated with predictions that involve internal ionizable residue that are important in processes like hydrogen transport and electron transfer. Furthermore, discrete water and ions cannot be modeled in implicit solvent, which are important in systems like membrane proteins and ion channels. We report on an explicit solvent constant pH molecular dynamics framework based on multi‐site λ‐dynamics (CPHMD^MSλD). In the CPHMD^MSλD framework, we performed seamless alchemical transitions between protonation and tautomeric states using multi‐site λ‐dynamics, and designed novel biasing potentials to ensure that the physical end‐states are predominantly sampled. We show that explicit solvent CPHMD^MSλD simulations model realistic pH‐dependent properties of proteins such as the Hen‐Egg White Lysozyme (HEWL), binding domain of 2‐oxoglutarate dehydrogenase (BBL) and N‐terminal domain of ribosomal protein L9 (NTL9), and the pK_a predictions are in excellent agreement with experimental values, with a RMSE ranging from 0.72 to 0.84 pK_a units. With the recent development of the explicit solvent CPHMD^MSλD framework for nucleic acids, accurate modeling of pH‐dependent properties of both major class of biomolecules—proteins and nucleic acids is now possible. Proteins 2014; 82:1319–1331. © 2013 Wiley Periodicals, Inc.