Subcellular communication through RNA transport and localized protein synthesis

Interest in the mechanisms of subcellular localization of mRNAs and the effects of localized translation has increased over the last decade. Polarized eukaryotic cells transport mRNA-protein complexes to subcellular sites, where translation of the mRNAs can be regulated by physiological stimuli. The long distances separating distal neuronal processes from their cell body have made neurons a useful model system for dissecting mechanisms of mRNA trafficking. Both the dendritic and axonal processes of neurons have been shown to have protein synthetic capacity and the diversity of mRNAs discovered in these processes continues to increase. Localized translation of mRNAs requires a co-ordinated effort by the cell body to target both mRNAs and necessary translational machinery into distal sites, as well as temporal control of individual mRNA translation. In addition to altering protein composition locally at the site of translation, some of the proteins generated in injured nerves retrogradely signal to the cell body, providing both temporal and spatial information on events occurring at distant subcellular sites.     

Analysis and design of RNA sequencing experiments for identifying isoform regulation

Through alternative splicing, most human genes express multiple isoforms that often differ in function. To infer isoform regulation from high-throughput sequencing of cDNA fragments (RNA-seq), we developed the mixture-of-isoforms (MISO) model, a statistical model that estimates expression of alternatively spliced exons and isoforms and assesses confidence in these estimates. Incorporation of mRNA fragment length distribution in paired-end RNA-seq greatly improved estimation of alternative-splicing levels. MISO also detects differentially regulated exons or isoforms. Application of MISO implicated the RNA splicing factor hnRNP H1 in the regulation of alternative cleavage and polyadenylation, a role that was supported by UV cross-linking-immunoprecipitation sequencing (CLIP-seq) analysis in human cells. Our results provide a probabilistic framework for RNA-seq analysis, give functional insights into pre-mRNA processing and yield guidelines for the optimal design of RNA-seq experiments for studies of gene and isoform expression.     

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.     

Quantitative measurement of Ca²(+) in the sarcoplasmic reticulum lumen of mammalian skeletal muscle

Skeletal muscle stores Ca²⁺ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²⁺ in the SR ([Ca²⁺]_SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²⁺]_SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²⁺]_{SR, free} is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²⁺]_{SR, free} to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²⁺ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²⁺ and for future studies of the role of SR Ca²⁺ in healthy and diseased mammalian muscle.     

NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

Endoplasmic reticulum (ER)-induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress-apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA-dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.     

Isolation of recombinant proteins from culture broth by co‐precipitation with an amino acid carrier to form stable dry powders

Protein‐coated microcrystals can be generated by co‐precipitation of protein and a water‐soluble crystalline carrier by addition to excess water miscible organic solvent. We have investigated this novel process for its utility in the concentration and partial purification of a recombinant protein exported into the culture broth during expression by Pichia pastoris. Co‐precipitation with a L ‐glutamine carrier selectively isolated the protein content of the culture broth, with a minimal number of steps, and simultaneously removed contaminants including a novel yeast metabolite. This pigment co‐elutes during aqueous chromatography but its elucidation as a benzoylated glycosamine suggested a simple route of removal by partition during the co‐precipitation process. Scale‐up of the process was readily achieved through in‐line mixing and subsequent reconstitution of the dried protein‐coated microcrystals yielded natively folded, bioactive protein. Additional washing of the crystals with saturated L ‐glutamine facilitated further purification of the recombinant protein immobilized on the L ‐glutamine carrier. Thus, we present a novel method for the harvesting of recombinant protein from culture broth as a dry powder, which may be of general applicability to bioprocessing. Biotechnol. Bioeng. 2010;106: 764–773. © 2010 Wiley Periodicals, Inc.     

Catch and measure–mass spectrometry‐based immunoassays in biomarker research

Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

When Did Carcharocles megalodon Become Extinct? A New Analysis of the Fossil Record

Carcharocles megalodon (“Megalodon”) is the largest shark that ever lived. Based on its distribution, dental morphology, and associated fauna, it has been suggested that this species was a cosmopolitan apex predator that fed on marine mammals from the middle Miocene to the Pliocene (15.9–2.6 Ma). Prevailing theory suggests that the extinction of apex predators affects ecosystem dynamics. Accordingly, knowing the time of extinction of C. megalodon is a fundamental step towards understanding the effects of such an event in ancient communities. However, the time of extinction of this important species has never been quantitatively assessed. Here, we synthesize the most recent records of C. megalodon from the literature and scientific collections and infer the date of its extinction by making a novel use of the Optimal Linear Estimation (OLE) model. Our results suggest that C. megalodon went extinct around 2.6 Ma. Furthermore, when contrasting our results with known ecological and macroevolutionary trends in marine mammals, it became evident that the modern composition and function of modern gigantic filter-feeding whales was established after the extinction of C. megalodon. Consequently, the study of the time of extinction of C. megalodon provides the basis to improve our understanding of the responses of marine species to the removal of apex predators, presenting a deep-time perspective for the conservation of modern ecosystems.