Elucidation of structure–function relationships in Methanocaldococcus jannaschii RNase P,a multi-subunit catalytic ribonucleoprotein

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.     

A polymetamorphic protein

Arc repressor is a homodimeric protein with a ribbon‐helix–helix fold. A single polar‐to‐hydrophobic substitution (N11L) at a solvent‐exposed position leads to population of an alternate dimeric fold in which 3₁₀ helices replace a β‐sheet. Here we find that the variant Q9V/N11L/R13V (S‐VLV), with two additional polar‐to‐hydrophobic surface mutations in the same β‐sheet, forms a highly stable, reversibly folded octamer with approximately half the?α‐helical content of wild‐type Arc. At low protein concentration and low ionic strength, S‐VLV also populates both dimeric topologies previously observed for N11L, as judged by NMR chemical shift comparisons. Thus, accumulation of simple hydrophobic mutations in Arc progressively reduces fold specificity, leading first to a sequence with two folds and then to a manifold bridge sequence with at least three different topologies. Residues 9–14 of S‐VLV form a highly hydrophobic stretch that is predicted to be amyloidogenic, but we do not observe aggregates of higher order than octamer. Increases in sequence hydrophobicity can promote amyloid aggregation but also exert broader and more complex effects on fold specificity. Altered native folds, changes in fold coupled to oligomerization, toxic pre‐amyloid oligomers, and amyloid fibrils may represent a near continuum of accessible alternatives in protein structure space.     

The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.     

Temporal integration in nasal lateralization and nasal detection of carbon dioxide

Two experiments examined time/concentration trading for the detection of carbon dioxide, an irritant with little or no odor. Experiment 1 employed the nasal lateralization method: subjects attempted to determine which nostril received carbon dioxide and which received pure air when presented simultaneously. Experiment 2 employed a temporal, two-alternative, forced-choice, detection paradigm with monorhinal stimulation. In both experiments, stimulus duration was varied at a number of fixed concentrations to determine the shortest, detectable pulse. Under both conditions, threshold pulse duration decreased as stimulus concentration increased. Power functions with exponents of less than negative one described the data quite well: More than a twofold increase in duration was needed to compensate for a twofold decrease in concentration. Thus, for carbon dioxide, the nasal trigeminal system functions as an imperfect integrator at threshold-level.     

Differential qualitative and temporal changes in the response of the hypothalamus-pituitary-adrenal axis in rats after localized or total-body irradiation

Stress such as exposure to ionizing radiation is able to activate the hypothalamus-pituitary-adrenal axis. The present study sought to examine the effects of different configurations of a 10-Gy gamma irradiation in rats on the hypothalamus-pituitary-adrenal axis to understand the mechanism of negative feedback by glucocorticoids induced by ionizing radiation. Specifically, we determined adrenocorticotropin and corticosterone levels in plasma as well as corticotrophin-releasing factor expression in the paraventricular nucleus of the hypothalamus by in situ hybridization from 6 h to 4 days after total-body, abdominal or head irradiation. In this study, we found an activation of the hypothalamus-pituitary-adrenal axis after radiation exposure. Plasma adrenocorticotropin and corticosterone levels were significantly increased after total-body and abdominal irradiation 3 days after exposure, in parallel with decreased labeling of corticotrophin-releasing factor mRNA in the parvocellular region of the paraventricular nucleus of the hypothalamus. Our results suggest that ionizing radiation activates the neuroendocrine system to protect the organism from the occurrence of radiation-induced inflammation.     

Negligible class II MHC presentation of B cell receptor-derived peptides by high density resting B cells

Resting B lymphocytes have been credited with inducing T cell tolerance to Ig-derived and monovalent self-Ags that are internalized via the B cell receptor (BCR). These conclusions are predicated upon the assumptions that resting B cells display BCR-associated peptides in class II MHC and that the cells remain quiescent during the course of experimental manipulation. To determine whether resting B cells display BCR-associated epitopes in class II MHC, we devised a sensitive assay that averted potential activation of B cells by Ag and minimized activation by prolonged culture. Ex vivo, Percoll-fractionated B cells expressing a kappa transgene encoding a T cell epitope were cultured with a reactive T cell hybridoma for 12 h. Whereas low density, LPS-activated, and BCR-activated B cells elicited significant IL-2 from the T cell hybridoma, resting high density B cells did not. Parallel results were obtained with normal B cells expressing a second epitope encoded by an endogenous V(H) gene. Anergic B cells, which are uniformly low density, also significantly stimulated the T cell hybridoma. Finally, longer culture periods with normal B cells resulted in a higher degree of B cell activation and significant stimulation of reactive T cell hybridomas. Our results provide evidence that activation of B cells profoundly enhances the processing and presentation of BCR-associated Ags.     

Susceptibility of staphylococci to natural and synthetic iron chelators

A total of 237 strains of staphylococci belonging to 27 species were tested for susceptibility to natural and synthetic iron-chelators. Coagulase-negative staphylococci were much more susceptible than coagulase-positive ones: 82 out of 148 coagulase-negative strains were susceptible to desferrioxamine B, rhodotorulic acid and ovotransferrin and 7 out of 89 coagulase-positive strains. A correlation between the susceptibility to iron-chelators species affiliation and the origin of strain was not found.     

The influence of intracellular idarubicin and daunorubicin levels on drug cytotoxicity in childhood acute leukemia

Uptake and efflux of two anthracyclines, idarubicin (IDA) and daunorubicin (DNR), was studied in childhood acute leukemia samples. A comparison of IDA and DNR transport phenomena in relation to drug cytotoxicity and expression of P-glycoprotein (PGP) was made. Intracellular content of IDA/DNR was determined by flow cytometry using the fluorescent properties of the drugs. In vitro drug cytotoxicity was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. PGP expression was analysed by flow cytometry. The uptake and efflux rates were non-significantly higher for IDA than DNR. There were no differences between three types of leukemia with respect to drug content during accumulation and retention. After correction for the cell volume, intracellular concentration of both drugs in each moment of uptake and efflux was significantly lower in relapsed ALL and AML samples in comparison with initial ALL cells. Efflux, but not uptake, of both drugs was inversely correlated with PGP expression and IDA, but not DNR, cytotoxicity. The cytotoxicity was correlated with drug accumulation for both drugs and with drug retention for IDA. In conclusion, it seems that (1) intracellular content was related to the lipophilic properties of the drugs rather than to the type of leukemia, (2) decreased intracellular concentration of both drugs might have an impact on compromised therapy results in AML and relapsed ALL children, (3) IDA presents higher cytotoxicity, which possibly might be decreased by the presence of PGP. These results might have a practical impact on the rational design of new chemotherapy protocols.