Translation is required for miRNA-dependent decay of endogenous transcripts.

Post-transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA-dependent repression. However, because transcript decay occurs co-translationally, whether target translation is a requirement for miRNA-dependent transcript destabilization remains unknown. To decouple these two molecular processes, we used cytosolic long noncoding RNAs (lncRNAs) as models for endogenous transcripts that are not translated. We show that, despite interacting with the miRNA-loaded RNA-induced silencing complex, the steady-state abundance and decay rates of these transcripts are minimally affected by miRNA loss. To further validate the apparent requirement of translation for miRNA-dependent decay, we fused two lncRNA candidates to the 3'-end of a protein-coding gene reporter and found this results in their miRNA-dependent destabilization. Further analysis revealed that the few natural lncRNAs whose levels are regulated by miRNAs in mESCs tend to associate with translating ribosomes, and possibly represent misannotated micropeptides, further substantiating the necessity of target translation for miRNA-dependent transcript decay. In summary, our analyses suggest that translation is required for miRNA-dependent transcript destabilization, and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.

Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides.

A modified 2'-deoxycytidine triphosphate derivative (dCTO TP) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dCTO TP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dCTO TP. When dCTO TP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.

Practical Antibody Recruiting by Metabolic Labeling with Caged Glycans.

We propose a de novo glycan display approach that combines metabolic labeling and a glycan-caging strategy as a facile editing method for cell-surface glycans. This method enables the introduction of antigen glycans onto cancer cells to induce immune responses through antibody recruiting. The caging strategy prevents the capture of α-rhamnose (an antigen glycan) by endogenous antibodies during the introduction of the glycan to the targeted cell surface, and subsequent uncaging successfully induces immune responses. Therefore, this study proposes a practical method for editing the cell-surface glycocalyx under promiscuous conditions, such as those in vivo, which paves the way for the development of glycan function analysis and regulation.

O-GlcNAcylation promotes fatty acid synthase activity under nutritional stress as a pro-survival mechanism in cancer cells.

Protein O-GlcNAcylation is a specific form of protein glycosylation that targets a wide range of proteins with important functions. O-GlcNAcylation is known to be deregulated in cancer and has been linked to multiple aspects of cancer pathology. Despite its ubiquity and importance, the current understanding of the role of O-GlcNAcylation in the stress response remains limited. In this study, we performed a quantitative chemical proteomics-based open study of the O-GlcNAcome in HeLa cells, and identified 163 differentially-glycosylated proteins under starvation, involving multiple metabolic pathways. Among them, fatty acid metabolism was found to be targeted and subsequent analysis confirmed that fatty acid synthase (FASN) is O-GlcNAcylated. O-GlcNAcylation led to enhanced de novo fatty acid synthesis (FAS) activity, and fatty acids contributed to the cytoprotective effects of O-GlcNAcylation under starvation. Moreover, dual inhibition of O-GlcNAcylation and FASN displayed a strong synergistic effect in vitro in inducing cell death in cancer cells. Together, the results from this study provide novel insights into the role of O-GlcNAcylation in the nutritional stress response and suggest the potential of combining inhibition of O-GlcNAcylation and FAS in cancer therapy.

Rainbow trout gastrointestinal mucus, mucin production, mucin glycosylation and response to lipopolysaccharide.

Mucus, whereof the highly glycosylated mucins are a major component, protects the epithelial mucosal surfaces. The aim of this study was to characterize the rainbow trout (Oncorhynchus mykiss) gastrointestinal mucus barrier function, mucin production, glycosylation and response to lipopolysaccharide. Both gastric and intestinal mucus was thick and impenetrable to bacteria-sized beads ex vivo. The secreted mucus covering the gastric epithelium predominantly contained sialylated mucins. Plume-like structures emerging from the gastric pits were both sialylated and fucosylated, indicating heterogeneity in gastric mucus secreted by the surface mucus cells and gland secretory cells, whereas intestinal mucus appeared more homogenous. In vivo metabolic mucin labelling revealed regional differences in mucin production and basal to apical transport, while lipopolysaccharide stimulation increased the rate of mucin production and basal to apical transport in both stomach and intestine. Using mass spectrometry, 34 mucin O-glycans were identified, with ∼70% of the relative abundance being sialylated, ∼40% di-sialylated and 20-25% fucosylated. No effects of lipopolysaccharide treatment were apparent regarding O-glycan repertoires, relative abundance of components, size distribution or core structures. Thus, the mucus production and organization differ between epithelial sites but provide a barrier to bacteria in both stomach and intestine. Furthermore, mucin production and basal to apical transport was stimulated by lipopolysaccharide in all regions, suggesting a mechanism to combat infections.

The caspase-like Gpi8 subunit of Candida albicans GPI transamidase is a metal-dependent endopeptidase.

GPI anchored proteins (GPI-APs) act at the frontiers of cells, decoding environmental cues and determining host-pathogen interactions in several lower eukaryotes. They are also essential for viability in lower eukaryotes. The GPI biosynthetic pathway begins at the ER and follows a roughly linear pathway to generate the complete precursor (CP) glycolipid. The GPI transamidase (GPIT) transfers this glycolipid to the C-terminal end of newly translated proteins after removing their GPI attachment signal sequence (SS). The GPIT subunit that cleaves SS is Gpi8, a protein with a conserved Cys/His catalytic dyad typical of cysteine proteases. A CaGPI8 heterozygous mutant accumulates CPs and has reduced cell surface GPI-APs. Using a simple cell-free assay, we demonstrate that the heterozygous CaGPI8 strain has low endopeptidase activity as well. The revertant strain is restored in all these phenotypes. CaGpi8 is also shown to be a metalloenzyme, whose protease activity is sensitive to agents that modify Cys/His residues.

Chemical Proteomic Profiling of Protein 4'-Phosphopantetheinylation in Mammalian Cells.

Protein 4'-phosphopantetheinylation is an essential post-translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as ß-alanine activation. To explore existing and new substrates of 4'-phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4'-phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4'-phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4'-phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).

Perfluoroaryl Azide Staudinger Reaction: A Fast and Bioorthogonal Reaction.

We report a fast Staudinger reaction between perfluoroaryl azides (PFAAs) and aryl phosphines, which occurs readily under ambient conditions. A rate constant as high as 18 m-1 s-1 was obtained between methyl 4-azido-2,3,5,6-tetrafluorobenzoate and methyl 2-(diphenylphosphanyl)benzoate in CD3 CN/D2 O. Furthermore, the iminophosphorane product was stable toward hydrolysis and aza-phosphonium ylide reactions. This PFAA Staudinger reaction proved to be an excellent bioothorgonal reaction. PFAA-derivatized mannosamine and galactosamine were successfully transformed into cell-surface glycans and efficiently labeled with phosphine-derivatized fluorophore-conjugated bovine serum albumin.

Expansion of metabolically labelled endocytic organelles and cytoskeletal cell structures in Giardia lamblia using optimised U-ExM protocols.

Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.

a6A-seq: N 6-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing.

The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics. The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs. Here we developed an a6A-seq method, which takes advantage of N6-allyladenosine (a6A) metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner. a6A plays a role as a chemical sequencing tag in that the iodination of a6A in mRNAs results in 1,N 6-cyclized adenosine (cyc-A), which induces base misincorporation during RNA reverse transcription, thus making a6A-labelled mRNAs detectable by sequencing. A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine. a6A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition. Compared with regular RNA-seq, a6A-seq could more sensitively detect the change of mRNA production over a time scale. The experiment of a6A-containing mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a6A-seq data. Together, our results show a6A-seq is an effective tool to study RNA dynamics.

Elucidating cell surface glycan imbalance through SERS guided metabolic glycan labelling: An appraisal of metastatic potential in cancer cells.

The intrinsic complexities of cell-surface glycans impede tracking the metabolic changes in cells. By coupling metabolic glycan labelling (MGL) and surface-enhanced Raman scattering (SERS), we employed the MGL-SERS strategy to elucidate the differential glycosylation pattern in cancer cell lines. Herein, for the first time, we are reporting an N-alkyl derivative of glucosamine (GlcNPhAlk) as a glycan labelling precursor. The extent of labelling was assessed by utilizing Raman imaging and verified by complementary fluorescence and Western blot analysis. MGL-SERS technique was implemented for a comparative evaluation of cell surface glycan imbalance in different cancer cells wherein a linear relationship between glycan expression and metastatic potential was established. Further, the effect of sialyltransferase inhibitor, P-3Fax-Neu5Ac, on metabolic labelling of GlcNPhAlk proved the incorporation of GlcNPhAlk to the terminal glycans through the sialic acid biosynthetic pathway. Hence, this methodology unveils the phenomenon of metastatic progression in cancer cells with inherent glycosylation-related dysplasia.

NanoSIMS observations of mouse retinal cells reveal strict metabolic controls on nitrogen turnover.

BACKGROUND: Most of the cells of the mammalian retina are terminally differentiated, and do not regenerate once fully developed. This implies that these cells have strict controls over their metabolic processes, including protein turnover. We report the use of metabolic labelling procedures and secondary ion mass spectrometry imaging to examine nitrogen turnover in retinal cells, with a focus on the outer nuclear layer, inner nuclear layer, and outer plexiform layer. RESULTS: We find that turnover can be observed in all cells imaged using NanoSIMS. However, the rate of turnover is not constant, but varies between different cellular types and cell regions. In the inner and outer nuclear layers, turnover rate is higher in the cytosol than in the nucleus of each cell. Turnover rates are also higher in the outer plexiform layer. An examination of retinal cells from mice that were isotopically labeled very early in embryonic development shows that proteins produced during this period can be found in all cells and cell regions up to 2 months after birth, even in regions of high turnover. CONCLUSIONS: Our results indicate that turnover in retinal cells is a highly regulated process, with strict metabolic controls. We also observe that turnover is several-fold higher in the synaptic layer than in cell layers. Nevertheless, embryonic proteins can still be found in this layer 2 months after birth, suggesting that stable structures persist within the synapses, which remain to be determined.

Tracking glycosylation in live cells using FTIR spectroscopy.

This is the first demonstration of the study of glycan protein turnover in living cells by FTIR with commercially available tetraacetylated N-Azidoacetyl-D-Mannosamine (Ac4ManNAz) label. The FTIR analysis has shown to be able to monitor the metabolism of glycans in living cells in real time. The method is simple, quantitative and requires equipment that are available in many laboratories. It can be used in a wide range of applications such as the study of glycosylation and cell-signalling.

Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum, Trichosurus vulpecula.

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum (Trichosurus vulpecula) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.

A modified gas-trapping method for high-throughput metabolic experiments in Drosophila melanogaster.

Metabolism is often studied in animal models, with the Drosophila melanogaster fruit fly model offering ease of genetic manipulation and high-throughput studies. Fly metabolism is typically studied using end-point assays that are simple but destructive, and do not provide information on the utilization of specific nutrients. To address these limitations, we adapted existing gas-trapping protocols to measure the oxidation of radiolabeled substrates (such as glucose) in multi-well plates. This protocol is cost effective, simple, and offers precise control over experimental diet and measurement time, thus being amenable to high-throughput studies. Furthermore, it is nondestructive, enabling time-course experiments and multiplexing with other parameters. Overall, this protocol is useful for merging fly genetics with metabolic studies to understand whole organism responses to different macronutrients.

The mitochondrial proteome of the moss Physcomitrella patens.

Extant basal land plants are routinely used to trace plant evolution and to track strategies for high abiotic stress resistance. Whereas the structure of mitochondrial genomes and RNA editing are already well studied, mitochondrial proteome research is restricted to a few data sets. While the mitochondrial proteome of the model moss Physcomitrella patens is covered to an estimated 15-25% by proteomic evidence to date, the available data have already provided insights into the evolution of metabolic compartmentation, dual targeting and mitochondrial heterogeneity. This review summarizes the current knowledge about the mitochondrial proteome of P. patens, and gives a perspective on its use as a mitochondrial model system. Its amenability to gene editing, metabolic labelling as well as fluorescence microscopy provides a unique platform to study open questions in mitochondrial biology, such as regulation of protein stability, responses to stress and connectivity to other organelles. Future challenges will include improving the proteomic resources for P. patens, and to link protein inventories and modifications as well as evolutionary differences to the functional level.

Glareosin: a novel sexually dimorphic urinary lipocalin in the bank vole, Myodes glareolus.

The urine of bank voles (Myodes glareolus) contains substantial quantities of a small protein that is expressed at much higher levels in males than females, and at higher levels in males in the breeding season. This protein was purified and completely sequenced at the protein level by mass spectrometry. Leucine/isoleucine ambiguity was completely resolved by metabolic labelling, monitoring the incorporation of dietary deuterated leucine into specific sites in the protein. The predicted mass of the sequenced protein was exactly consonant with the mass of the protein measured in bank vole urine samples, correcting for the formation of two disulfide bonds. The sequence of the protein revealed that it was a lipocalin related to aphrodisin and other odorant-binding proteins (OBPs), but differed from all OBPs previously described. The pattern of secretion in urine used for scent marking by male bank voles, and the similarity to other lipocalins used as chemical signals in rodents, suggest that this protein plays a role in male sexual and/or competitive communication. We propose the name glareosin for this novel protein to reflect the origin of the protein and to emphasize the distinction from known OBPs.

Measuring Transcription Rate Changes via Time-Course 4-Thiouridine Pulse-Labelling Improves Transcriptional Target Identification.

Identifying changes in the transcriptional regulation of target genes from high-throughput studies is important for unravelling molecular mechanisms controlled by a given perturbation. When measuring global transcript levels only, the effect of the perturbation [e.g., transcription factor (TF) overexpression or drug treatment] on its target genes is often obscured by delayed feedback and secondary effects until the changes are fully propagated. As a proof of principle, we show that selective measuring of transcripts that are only synthesised after a perturbation [4-thiouridine (4sU) sequencing (4sU-seq)] is a more sensitive method to identify targets and time-dependent transcriptional responses than global transcript profiling. By metabolically labelling RNA in a time-course setup, we could vastly increase the sensitivity of MYCN target gene detection compared to traditional RNA sequencing. The validity of targets identified by 4sU-seq was demonstrated using chromatin immunoprecipitation sequencing and neuroblastoma microarray tumour data. Here, we describe the methodology, both molecular biology and computational aspects, required to successfully apply this 4sU-seq approach.