In germ cells, Piwi proteins interact with a specific class of small noncoding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. Basic models describe the overall operation of piRNA pathways. However, the protein compositions of Piwi complexes, the critical protein-protein interactions that drive small RNA production and target recognition, and the precise molecular consequences of conserved localization to germline structures, call nuage, remains poorly understood. We purified the three murine Piwi family proteins, MILI, MIWI, and MIWI2, from mouse germ cells and characterized their interacting protein partners. Piwi proteins were found in complex with PRMT5/ WDR77, an enzyme that dimethylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, we found that Piwi proteins are arginine methylated at conserved positions in their N termini. These modifications are essential to direct complex formation with specific members of the Tudor protein family. Recognition of methylarginine marks by Tudor proteins can drive the localization of Piwi proteins to cytoplasmic foci in an artificial setting, supporting a role for this interaction in Piwi localization to nuage, a characteristic that correlates with proper operation of the piRNA pathway and transposon silencing in multiple organisms.[Keywords: Arginine methyation; piRNAs; transposon silencing; tudor proteins] Supplemental material is available at http://www.genesdev.org.

Bioorthogonal chemistry provides an exciting new strategy to visualize protein expression, track protein localization, measure protein activity, and identify protein interaction partners in living systems.[1] Two steps are typically involved in this approach: 1) the incorporation of a bioorthogonal group into a protein through either a biochemical pathway or semisynthesis; 2) a site-specific reaction between the protein that carries the bioorthogonal group and a cognate small-molecule probe. Although a plethora of methods have been developed to address the first step, such as non-sense suppression mutagenesis, [2] expressed protein ligation, [3] metabolic engineering, [4] and tagging-via-substrate, [5] only a small number of bioorthogonal reactions are known for the second step. These site-specific reactions include the acid-catalyzed nucleophilic addition of hydrazine to a ketone or aldehyde, [6] Staudinger ligation, [7] Cu I -catalyzed azide-alkyne 1,3-dipolar cycloaddition (click chemistry), [8] strain-promoted azide-alkyne 1,3-dipolar cycloaddition, [9] and the oxidative coupling of aniline.[10] To fully realize the potential of bioorthogonal chemistry in probing protein function, there is an urgent need for the discovery of additional bioorthogonal reactions with robust reaction attributes. Herein, we report a bioorthogonal, photoinducible 1,3-dipolar cycloaddition reaction that allows rapid and highly selective modification of proteins carrying a diaryl tetrazole group in biological media.Forty years ago, Huisgen and co-workers reported a photoactivated 1,3-dipolar cycloaddition reaction between 2,5-diphenyltetrazole and methyl crotonate.[11] A concerted reaction mechanism was proposed, whereby the diaryl tetrazole undergoes a facile cycloreversion reaction upon photoirradiation to release N 2 and generate in situ a nitrile imine dipole, which cyclizes spontaneously with an alkene dipolarophile to afford a pyrazoline cycloadduct (Scheme 1). The photolysis of diaryl tetrazoles was found to be extremely efficient upon UV irradiation at 290 nm, with quantum yields in the range 0.5-0.9.[12] Despite its robust mechanism, this photoactivated reaction has seen very few applications in the past four decades.[13]In our initial studies, we identified an extremely mild photoactivation procedure in the use of a hand-held UV lamp from UVP (UVM-57, 302 nm, 115 V, 0.16 amps). Under these mild conditions, the solvent compatibility, functional-group tolerance, regioselectivity, and yield of the photoactivated 1,3-dipolar cycloaddition reaction were excellent. [14] We then examined the reaction kinetics by incubating a tetrazole peptide with acrylamide in phosphate-buffered saline (PBS) at pH 7.5 under UV light (302 nm; see Figure S1 in the Supporting Information). We found that the photolysis of the tetrazole peptide to generate the nitrile imine intermediate was extremely rapid, with a first-order rate constant k 1 = 0.14 s À1 ; the subsequent cycloaddition with the dipolarophile acrylamide proceeded very efficiently, w...

We report a tetrazole-based, photoclick chemistry that can be employed to selectively functionalize an alkene genetically encoded in a protein inside E. coli cells. The reaction involved the treatment of E. coli cells with cell-permeable tetrazoles followed by a brief photo irradiation at 302 nm (4 min) and an overnight incubation at 4 degrees C. This in vivo alkene functionalization procedure was simple, straightforward, and nontoxic to E. coli cells. Additionally, fluorescent adducts were formed, facilitating the monitoring of the reaction in vivo. This reaction should offer a new tool for the study of alkene-containing proteins in living systems.

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/ingel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were

Intraflagellar transport proteins (IFT) are required for hedgehog (Hh) signalling transduction that is essential for bone development, however, how IFT proteins regulate Hh signalling in osteoblasts (OBs) remains unclear. Here we show that deletion of ciliary IFT80 in OB precursor cells (OPC) in mice results in growth retardation and markedly decreased bone mass with impaired OB differentiation. Loss of IFT80 blocks canonical Hh–Gli signalling via disrupting Smo ciliary localization, but elevates non-canonical Hh–Gαi–RhoA–stress fibre signalling by increasing Smo and Gαi binding. Inhibition of RhoA and ROCK activity partially restores osteogenic differentiation of IFT80-deficient OPCs by inhibiting non-canonical Hh–RhoA–Cofilin/MLC2 signalling. Cytochalasin D, an actin destabilizer, dramatically restores OB differentiation of IFT80-deficient OPCs by disrupting actin stress fibres and promoting cilia formation and Hh–Gli signalling. These findings reveal that IFT80 is required for OB differentiation by balancing between canonical Hh–Gli and non-canonical Hh–Gαi–RhoA pathways and highlight IFT80 as a therapeutic target for craniofacial and skeletal abnormalities.

AIM:To investigate the effect of omega-3 fatty acid parenteral supplementation postoperatively on clinical outcomes and immunomodulation in colorectal cancer patients. METHODS:Forty-two patients undergoing radical colorectal cancer resection with an indication for total parenteral nutrition postoperatively were enrolled in this prospective, double-blind, randomized, controlled study. Patients received total parenteral nutrition supplemented with either soybean oil (LCT; Intralipid CONCLUSION:Postoperative supplementation of omega-3 fatty acids may have a favorable effect on the outcomes in colorectal cancer patients undergoing radical resection by lowering the magnitude of inflammatory responses and modulating the immune response.

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a wellcontrolled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age-and sex-matched control rats (n ‫؍‬ 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ϳ40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ioncurrent data (e.g. signal-to-noise ratio > 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS. Molecular & Cellular Proteomics 12:

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.