pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains.

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

Replication Protein A Phosphorylation Facilitates RAD52-Dependent Homologous Recombination in BRCA-Deficient Cells.

Loss of RAD52 is synthetically lethal in BRCA-deficient cells, owing to its role in backup homologous recombination (HR) repair of DNA double-strand breaks (DSBs). In HR in mammalian cells, DSBs are processed to single-stranded DNA (ssDNA) overhangs, which are then bound by replication protein A (RPA). RPA is exchanged for RAD51 by mediator proteins: in mammals, BRCA2 is the primary mediator; however, RAD52 provides an alternative mediator pathway in BRCA-deficient cells. RAD51 stimulates strand exchange between homologous DNA duplexes, a critical step in HR. RPA phosphorylation and dephosphorylation are important for HR, but its effect on RAD52 mediator function is unknown. Here, we show that RPA phosphorylation is required for RAD52 to salvage HR in BRCA-deficient cells. In BRCA2-depleted human cells, in which the only available mediator pathway is RAD52 dependent, the expression of a phosphorylation-deficient RPA mutant reduced HR. Furthermore, RPA-phosphomutant cells showed reduced association of RAD52 with RAD51. Interestingly, there was no effect of RPA phosphorylation on RAD52 recruitment to repair foci. Finally, we show that RPA phosphorylation does not affect RAD52-dependent ssDNA annealing. Thus, although RAD52 can be recruited independently of RPA's phosphorylation status, RPA phosphorylation is required for RAD52's association with RAD51 and its subsequent promotion of RAD52-mediated HR.

Identification of Potent and Selective Inhibitors of Fat Mass Obesity-Associated Protein Using a Fragment-Merging Approach.

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO-selective inhibitors.

More dynamic, more quantitative, unexpectedly intricate: Advanced understanding on synaptic RNA localization in learning and memory.

Synaptic signaling exhibits great diversity, complexity, and plasticity which necessitates maintenance and rapid modification of a local proteome. One solution neurons actively exploit to meet such demands is the strategic deposition of mRNAs encoding proteins for both basal and experience-driven activities into ribonucleoprotein complexes at the synapse. Transcripts localized in this manner can be rapidly accessed for translation in response to a diverse range of stimuli in a temporal- and spatially-restricted manner. Here we review recent findings on localized RNAs and RNA binding proteins in the context of learning and memory, as revealed by cutting-edge in-vitro and in-vivo technologies capable of yielding quantitative and dynamic information. The new technologies include proteomic and transcriptomic analyses, high-resolution multiplexed RNA imaging, single-molecule RNA tracking in living neurons, animal models and human neuron cell models. Among many recent advances in the field, RNA chemical modification has emerged as one of the new regulatory layers of gene expression at synapse that is complex and yet largely unexplored. These exciting new discoveries have enhanced our understanding of the modulation mechanisms of synaptic gene expression and their roles in cognition.

BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication.

BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1-BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.

BRCA1 and BRCA2: different roles in a common pathway of genome protection.

The proteins encoded by the two major breast cancer susceptibility genes, BRCA1 and BRCA2, work in a common pathway of genome protection. However, the two proteins work at different stages in the DNA damage response (DDR) and in DNA repair. BRCA1 is a pleiotropic DDR protein that functions in both checkpoint activation and DNA repair, whereas BRCA2 is a mediator of the core mechanism of homologous recombination. The links between the two proteins are not well understood, but they must exist to explain the marked similarity of human cancer susceptibility that arises with germline mutations in these genes. As discussed here, the proteins work in concert to protect the genome from double-strand DNA damage during DNA replication.

A birth-to-death view of mRNA from the RNA recognition motif perspective.

RNA binding proteins are a large and varied group of factors that are the driving force behind post-transcriptional gene regulation. By analogy with transcription factors, RNA binding proteins bind to various regions of the mRNAs that they regulate, usually upstream or downstream from the coding region, and modulate one of the five major processes in mRNA metabolism: splicing, polyadenylation, export, translation and decay. The most abundant RNA binding protein domain is called the RNA Recognition Motif (RRM)1. It is probably safe to say that an RRM-containing protein is making some contact with an mRNA throughout its existence. The transcriptional counterpart would likely be the histones, yet the multitude of specific functions that are results of RRM based interactions belies the universality of the motif. This complex and diverse application of a single protein motif was used as the basis to develop an advanced graduate level seminar course in RNA:protein interactions. The course, utilizing a learner-centered empowerment model, was developed to dissect each step in RNA metabolism from the perspective of an RRM containing protein. This provided a framework to discuss the development of specificity for the RRM for each required process.

Bacterial bioreactors for high yield production of recombinant protein.

We developed a new bacterial expression system that utilizes a combination of attributes (low temperature, induction of an mRNA-specific endoribonuclease causing host cell growth arrest, and culture condensation) to facilitate stable, high level protein expression, almost 30% of total cellular protein, without background protein synthesis. With the use of an optimized vector, exponentially growing cultures could be condensed 40-fold without affecting protein yields, which lowered sample labeling costs to a few percent of the cost of a typical labeling experiment. Because the host cells were completely growth-arrested, toxic amino acids such as selenomethionine and fluorophenylalanine were efficiently incorporated into recombinant proteins in the absence of cytotoxicity. Therefore, this expression system using Escherichia coli as a bioreactor is especially well suited to structural genomics, large-scale protein expressions, and the production of cytotoxic proteins.

Pharmacokinetics, tissue residue and plasma protein binding of ofloxacin in goats.

Ofloxacin was administered to six male goats intravenously (5 mg/kg) to determine its kinetic behavior, tissue residue, in vitro plasma protein binding and to compute a rational dosage regimen. The concentration of ofloxacin in plasma and tissue samples collected at prescheduled time were estimated by using HPLC. The pharmacokinetic parameters were determined by non-compartmental model and plasma protein binding was estimated by equilibrium dialysis technique. The therapeutic concentration (> or =0.5 microg/ml) was maintained up to 36 h and the initial concentration at 2.5 min (14.76 +/- 0.47 microg/ml) declined to 0.05 +/- 0.03 microg/ml at 96 h with a secondary peak (0.64 +/- 0.15 microg/ml) at 24 h. The mean AUC, AUMC, t1/2, MRT, Cl and Vd were calculated to be 58.94 +/- 19.43 microg x h/ml, 1539.57 +/- 724.69 microg x h2/ml, 15.58 +/- 1.87 h, 22.46 +/- 2.71 h, 135.60 +/- 31.12 ml/h/kg and 2.85 +/- 0.74 L/kg respectively. Significantly high concentration of drug was detected in different tissues after 24 h of intravenous dosing of 5 mg/kg, at 24 h interval for 5 days. The in vitro plasma protein binding of ofloxacin was found to be 15.28 +/- 0.94%. Based on these kinetic parameters, a loading dose of 5 mg/kg followed by the maintenance dose of 3 mg/kg at 24 h dosing interval by intravenous route is recommended.