Beyond the Western front:targeted proteomics and organelle abundance profiling

The application of westerns or immunoblotting techniques for assessing the composition, dynamics, and purity of protein extracts from plant material has become common practice. While the approach is reproducible, can be readily applied and is generally considered robust, the field of plant science suffers from a lack of antibody variety against plant proteins. The development of approaches that employ mass spectrometry to enable both relative and absolute quantification of many hundreds of proteins in a single sample from a single analysis provides a mechanism to overcome the expensive impediment in having to develop antibodies in plant science. We consider it an opportune moment to consider and better develop the adoption of multiple reaction monitoring (MRM)-based analyses in plant biochemistry

The<i> Arabidopsis</i> cytosolic proteome:the metabolic heart of the cell

The plant cytosol is the major intracellular fluid that acts as the medium for inter-organellar crosstalk and where a plethora of important biological reactions take place. These include its involvement in protein synthesis and degradation, stress response signaling, carbon metabolism, biosynthesis of secondary metabolites, and accumulation of enzymes for defense and detoxification. This central role is highlighted by estimates indicating that the majority of eukaryotic proteins are cytosolic. Arabidopsis thaliana has been the subject of numerous proteomic studies on its different subcellular compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted proteomic characterizations of complexes is included. Despite this, few groups are currently applying advanced proteomic approaches to this important metabolic space. This review will highlight the current state of the Arabidopsis cytosolic proteome since its initial characterization a few years ago

An NF-κB–Dependent Role for JunB in the Induction of Proinflammatory Cytokines in LPS-Activated Bone Marrow–Derived Dendritic Cells

BACKGROUND: Dendritic cells (DCs) play a key role in the induction of adaptive and memory immune responses. Upon encounter with pathogens, they undergo a complex maturation process and migrate toward lymphoid organs where they stimulate immune effector cells. This process is associated with dramatic transcriptome changes, pointing to a paramount role for transcription factors in DC activation and function. The regulation and the role of these transcription factors are however ill-defined and require characterization. Among those, AP-1 is a family of dimeric transcription complexes with an acknowledged role in the control of immunity. However, it has not been studied in detail in DCs yet. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have investigated the regulation and function of one of its essential components, JunB, in primary bone marrow-derived DCs induced to maturate upon stimulation by Escherichia coli lipopolysaccharide (LPS). Our data show fast and transient NF-kappaB-dependent transcriptional induction of the junb gene correlating with the induction of the TNFalpha, IL-6, and IL-12 proinflammatory cytokines. Inhibition of JunB protein induction by RNA interference hampered the transcriptional activation of the TNF-alpha, IL-6, and IL-12p40 genes. Consistently, chromatin immunoprecipitation experiments showed LPS-inducible binding of JunB at AP-1-responsive sites found in promoter regions of these genes. Concomitant LPS-inducible NF-kappaB/p65 binding to these promoters was also observed. CONCLUSIONS/SIGNIFICANCE: We identified a novel role for JunB--that is, induction of proinflammatory cytokines in LPS-activated primary DCs with NF-kappaB acting not only as an inducer of JunB, but also as its transcriptional partner

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo

Multiple marker abundance profiling:combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live)

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp(4), serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-β1,4Araf-β1,2Araf-β1,2Araf) side chains in an α-linkage, to yield Hyp-Araf(4) which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its’ product, Hyp-Araf(4), point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae