Spectral editing: selection of methyl groups in multidimensional solid-state magic-angle spinning NMR

A simple spectroscopic filtering technique is presented that may aid the assignment of ¹³C and ¹⁵N resonances of methyl-containing amino-acids in solid-state magic-angle spinning (MAS) NMR. A filtering block that selects methyl resonances is introduced in two-dimensional (2D) ¹³C-homonuclear and ¹⁵N–¹³C heteronuclear correlation experiments. The 2D ¹³C–¹³C correlation spectra are recorded with the methyl filter implemented prior to a ¹³C–¹³C mixing step. It is shown that these methyl-filtered ¹³C-homonuclear correlation spectra are instrumental in the assignment of C_δ resonances of leucines by suppression of C_γ–C_δ cross peaks. Further, a methyl filter is implemented prior to a ¹⁵N–¹³C transferred-echo double resonance (TEDOR) exchange scheme to obtain 2D ¹⁵N–¹³C heteronuclear correlation spectra. These experiments provide correlations between methyl groups and backbone amides. Some of the observed sequential ¹⁵N–¹³C correlations form the basis for initial sequence-specific assignments of backbone signals of the outer-membrane protein G.     

Proteomics for biodeterioration of wood (Pinus taeda L.): Challenging analysis by 2-D PAGE and MALDI-TOF/TOF/MS

Proteins expressed by the brown-rot fungus Gloeophyllum trabeum were characterized from inoculated southern yellow pine sapwood undergoing decay, from pure cultures of the fungus and from uninoculated pinewood. Analysis was carried out by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF/TOF/MS. No proteins were detected from the clean uncontaminated wood. The inoculated wood undergoing active brown-rot decay produced 76 proteins, including the Fenton-chemistry related enzymes, alcohol oxidase, lipoxygenase, and catalase. One hundred and eleven proteins were detected from the pure culture and most were common metabolic proteins. A majority of proteins in both samples were identified as hypothetical proteins. A surprising result is that there was very little overlap between proteins found in both sets of samples, indicating a very different mechanism in action when the fungus is growing on a cellulose-based nutrient source (wood) versus glucose media. This study also highlights a current limitation of this approach, which is the limited protein and genomic sequence information annotated on the public databases. Of the 187 proteins characterized, only 36 were identified with confidence. To our knowledge, this is the first reported proteomic analysis of pinewood decayed by a brown-rot fungus and provides the initial characterization of proteins involved in this type of wood biodeterioration. Although significant limitations still exist in identifying the proteins, this limitation will diminish as functional proteins are identified and added to the databases.     

Effects of Chronic Restraint Stress and 17-β-Estradiol Replacement on Oxidative Stress in the Spinal Cord of Ovariectomized Female Rats

Previous studies have shown sex-specific oxidative changes in spinal cord of rats submitted to chronic stress, which may be due to gonadal hormones. Here, we assessed total radical-trapping potential (TRAP), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and lipid peroxidation (evaluated by the TBARS test) in the spinal cord of ovariectomized (OVX) female rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided into controls and chronically stressed (for 40 days). Our findings demonstrate that chronic stress decreased TRAP, and increased SOD activity in spinal cord homogenates from ovariectomized female rats and had no effect on GPx activity. On the other hand, groups receiving 17β-estradiol replacement presented a decreased GPx activity, but no alteration in TRAP and in SOD activity. No differences in the TBARS test were found in any of the groups analyzed. In conclusion, our results support the idea that chronic stress induces an imbalance between SOD and GPx activities, additionally decreasing TRAP. Estradiol replacement did not reverse the effects of chronic stress, but induced a decrease in GPx activity. Therefore, estradiol replacement in ovariectomized chronically stressed rats could make the spinal cord more susceptible to oxidative injury.     

pK(a) values for the unfolded state under native conditions explain the pH-dependent stability of PGB1

Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK_a values. However, pK_a values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK_a values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK_a values were determined for carboxyl groups by monitoring their pH-dependent ¹³C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK_a values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK_a values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK_a values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.     

Inducible Priming Phosphorylation Promotes Ligand-independent Degradation of the IFNAR1 Chain of Type I Interferon Receptor

Phosphorylation-dependent ubiquitination and ensuing down-regulation and lysosomal degradation of the interferon α/β receptor chain 1 (IFNAR1) of the receptor for Type I interferons play important roles in limiting the cellular responses to these cytokines. These events could be stimulated either by the ligands (in a Janus kinase-dependent manner) or by unfolded protein response (UPR) inducers including viral infection (in a manner dependent on the activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and -independent pathways converge on phosphorylation of Ser⁵³⁵ within the IFNAR1 degron leading to recruitment of β-Trcp E3 ubiquitin ligase and concomitant ubiquitination and degradation. Casein kinase 1α (CK1α) was shown to directly phosphorylate Ser⁵³⁵ within the ligand-independent pathway. Yet given the constitutive activity of CK1α, it remained unclear how this pathway is stimulated by UPR. Here we report that induction of UPR promotes the phosphorylation of a proximal residue, Ser⁵³², in a pancreatic endoplasmic reticulum kinase-dependent manner. This serine serves as a priming site that promotes subsequent phosphorylation of IFNAR1 within its degron by CK1α. These events play an important role in regulating ubiquitination and degradation of IFNAR1 as well as the extent of Type I interferon signaling.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

Expression and a role of functionally coupled P2Y receptors in human dendritic cells

We investigated the physiology and function of P2Y receptors expressed in human dendritic cells (DCs) differentiated in vitro from CD14+ cells (DC-14). These were obtained after a 10 day stimulation period in GM-CSF, IL-4 and monocyte conditioned medium. DC-14 were found to express high amounts of MHC class II, B7, CD40 as well as CD83. The functional analysis, using single cell Ca2+ imaging, demonstrated the expression of at least three subtypes of P2Y receptors. We further found using patch-clamp measurements that ATP evoked a pertussis toxin insensitive non-selective cation current with a peak current amplitude of -276+/-43 pA (holding potential -80 mV, n = 23). This current was not Ca(2+)-activated, since it was still observed under conditions of high intracellular Ca2+ buffering and could be blocked by Gd3+ (0.5 mM). In addition, intracellular application of GTP-gamma-S (0.3 mM) also activated the current. Interestingly, DC-14 redirected the orientation of their dendrites as well as cell shape towards a pipette containing ATP as observed with time lapse microscopy. These data suggest that in human DCs, ATP acts via P2Y receptors and induces chemokine effects.     

Mitochondrial Uncoupling: Role of Uncoupling Protein Anion Carriers and Relationship to Thermogenesis and Weight Control “The Benefits of Losing Control”

Uncoupling proteins, a subgroup of the mitochondrial anion transporter superfamily, have beenidentified in prokaryotes, plants, and mammalian cells. Evolutionary conservation of thesemolecules reflects their importance as regulators of two critical mitochondrial functions, i.e.,ATP synthesis and the production of reactive oxygen species (ROS). Although the amino acidsequences of the three mammalian uncoupling proteins, UCP1, UCP2 and UCP3, are verysimilar, each homolog is the product of a unique gene and important differences have beendemonstrated in their tissue-specific expression and regulation. UCP1 and UCP3 appear to bekey regulators of energy expenditure, and hence, nonshivering thermogenesis, either in brownadipose tissue (UCP1) or skeletal muscle (UCP3). UCP2 is expressed more ubiquitously,although generally at low levels, in many tissues. There is conflicting evidence about itsimportance as a regulator of resting metabolic rate. However, evidence suggests that thishomolog might modulate the mitochondrial generation of ROS in some cell types, includingmacrophages and hepatocytes. While the induction of various uncoupling protein homologsprovides adaptive advantages, both to the organism (e.g., thermogenesis) and to individual cells(e.g., reduced ROS), increased uncoupling protein activity also increases cellular vulnerability tonecrosis by compromising the mitochondrial membrane potential. This narrow risk—benefitmargin necessitates tight control of uncoupling protein activity in order to preserve cellularviability and much remains to be learned about the regulatory mechanisms involved.