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.     

The SEP domain of p47 acts as a reversible competitive inhibitor of cathepsin L

The solution structure of the human p47 SEP domain in a construct comprising residues G1-S2-p47(171-270) was determined by NMR spectroscopy. A structure-derived hypothesis about the domains' function was formulated and pursued in binding experiments with cysteine proteases. The SEP domain was found to be a reversible competitive inhibitor of cathepsin L with a K_i of 1.5 μM. The binding of G1-S2-p47(171-270) to cathepsin L was mapped by biochemical assays and the binding interface was investigated by NMR chemical shift perturbation experiments.     

The cyclin D1-dependent kinase associates with the pre-replication complex and modulates RB.MCM7 binding

The capacity of the cyclin D-dependent kinase to promote G(1) progression through modulation of RB.E2F is well documented. We now demonstrate that the cyclin D1/CDK4 kinase binds to components of the MCM complex. MCM7 and MCM3 were identified as cyclin D1-binding proteins. Catalytically active cyclin D1/CDK4 complexes were incorporated into chromatin-bound protein complexes with the same kinetics as MCM7 and MCM3, where they associated specifically with MCM7. Although the cyclin D1-dependent kinase did not phosphorylate MCM7, active cyclin D1/CDK4, but not cyclin E/CDK2, did catalyze the dissociation of an RB.MCM7 complex. Finally, expression of an active D1/CDK4 kinase but not cyclin E/CDK2 promoted the removal of RB from chromatin-bound protein complexes. Our data suggest that D1/CDK4 complexes play a direct role in altering an inhibitory RB.MCM7 complex possibly allowing for setting of the origin in preparation for DNA replication.     

Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme

The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.