Proteomic analysis of Artemisia annua – towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328–331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria. Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/ viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin. Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin’s biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703

MS-based clinical proteomics: biomarker discovery in men’s cancer

Importance of biomarker discovery in men’s cancer diagnosis and prognosis Each year around 10,000 men in the UK die as a result of prostate cancer (PCa) making it the 3rd most common cancer behind lung and breast cancer; worldwide more than 670,000 men are diagnosed every year with the disease [1]. Current methods of diagnosis of PCa mainly rely on the detection of elevated prostate-specific antigen (PSA) levels in serum and/or physical examination by a doctor for the detection of an abnormal prostate. PSA is a glycoprotein produced almost exclusively by the epithelial cells of the prostate gland [2]. Its role is not fully understood, although it is known that it forms part of the ejaculate and its function is to solubilise the sperm to give them the mobility to swim. Raised PSA levels in serum are thought to be due to both an increased production of PSA from the proliferated prostate cells, and a diminished architecture of affected cells, allowing an easier distribution of PSA into the wider circulatory system

Discovery and Longitudinal Evaluation of Candidate Protein Biomarkers for Disease Recurrence in Prostate Cancer

When compared with hormonal therapy alone, treatment with combined hormone and radiation therapy (CHRT) gives improved disease-specific survival outcomes for patients with prostate cancer; however, a significant number of CHRT patients still succumb to recurrent disease. The purpose of this study was to use longitudinal patient samples obtained as part of an ongoing noninterventional clinical trial (ICORG06-15) to identify and evaluate a potential serum protein signature of disease recurrence. Label-free LC–MS/MS based protein discovery was undertaken on depleted serum samples from CHRT patients who showed evidence of disease recurrence (<i>n</i> = 3) and time-matched patient controls (<i>n</i> = 3). A total of 104 proteins showed a significant change between these two groups. Multiple reaction monitoring (MRM) assays were designed for a subset of these proteins as part of a panel of putative prostate cancer biomarkers (41 proteins) for evaluation in longitudinal serum samples. These data revealed significant interpatient variability in individual protein expression between time of diagnosis, disease recurrence, and beyond and serve to highlight the importance of longitudinal patient samples for evaluating the use of candidate protein biomarkers in disease monitoring

Janus PEG-Based Dendrimers for Use in Combination Therapy: Controlled Multi-Drug Loading and Sequential Release

The increasing use of drug combinations to treat disease states, such as cancer, calls for improved delivery systems that are able to deliver multiple agents. Herein, we report a series of novel Janus dendrimers with potential for use in combination therapy. Different generations (first and second) of PEG-based dendrons containing two different “model drugs”, benzyl alcohol (BA) and 3-phenylpropionic acid (PPA), were synthesized. BA and PPA were attached via two different linkers (carbonate and ester, respectively) to promote differential drug release. The four dendrons were coupled together via (3 + 2) cycloaddition chemistries to afford four Janus dendrimers, which contained varying amounts and different ratios of BA and PPA, namely, <b>(BA)</b>_<b>2</b><b>-G1-G1-(PPA)</b>_<b>2</b>, <b>(BA)</b>_<b>4</b><b>-G2-G1-(PPA)</b>_<b>2</b>, <b>(BA)</b>_<b>2</b><b>-G1-G2-(PPA)</b>_<b>4</b>, and <b>(BA)</b>_<b>4</b><b>-G2-G2-(PPA)</b>_<b>4</b>. Release studies in plasma showed that the dendrimers provided sequential release of the two model drugs, with BA being released faster than PPA from all of the dendrons. The different dendrimers allowed delivery of increasing amounts (0.15–0.30 mM) and in exact molecular ratios (1:2; 2:1; 1:2; 2:2) of the two model drug compounds. The dendrimers were noncytotoxic (100% viability at 1 mg/mL) toward human umbilical vein endothelial cells (HUVEC) and nontoxic toward red blood cells, as confirmed by hemolysis studies. These studies demonstrate that these Janus PEG-based dendrimers offer great potential for the delivery of drugs via combination therapy

Regulation of Toll-like Receptor Signaling by the SF3a mRNA Splicing Complex

<div><p>The innate immune response plays a key role in fighting infection by activating inflammation and stimulating the adaptive immune response. However, chronic activation of innate immunity can contribute to the pathogenesis of many diseases with an inflammatory component. Thus, various negatively acting factors turn off innate immunity subsequent to its activation to ensure that inflammation is self-limiting and to prevent inflammatory disease. These negatively acting pathways include the production of inhibitory acting alternate proteins encoded by alternative mRNA splice forms of genes in Toll-like receptor (TLR) signaling pathways. We previously found that the SF3a mRNA splicing complex was required for a robust innate immune response; SF3a acts to promote inflammation in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3a1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3a in murine macrophages. Surprisingly, in macrophages, SF3a has significant preference for mRNA splicing events within innate immune signaling pathways compared with other biological pathways, thereby affecting the splicing of specific genes in the TLR signaling pathway to modulate the innate immune response.</p></div

SF3a1 inhibition leads to intron retention in several TLR signaling pathway genes.

<p>(A,B) These panels depict sequence reads (average of 3 replicates, each; generated by DEXSeq) across IRAK1 or IKKβ (5’ end of gene on right, control siRNA in red, SF3a1 siRNA in blue). The retained introns (intron 1 in IRAK1 or intron 15 in IKKβ) are shaded in purple. (C-I) These panels display the results of qPCR assays on RAW264.7 cells used to monitor production of the indicated mRNA isoforms (expression normalized so that 1 is the expression in the presence of control siRNA). CT indicates control siRNA in this an all other figures. LPS exposures were performed for six hours in the presence of 20 ng/ml LPS. Asterisks indicate results that were statistically different than control in this and all other figures.</p

Multiple genes in the TLR signaling pathway mediate the effects of SF3a1 on innate immunity.

<p>RAW264.7 macrophages were treated with the indicated siRNAs or control non-targeting siRNA and subsequently exposed to 20 ng/ml LPS for 6 hours. IL-6 production was monitored by ELISA. In panels B and C, cells were treated with multiple siRNAs simultaneously, as indicated; in panel A, only a single siRNA was used in each case.</p

SF3a1 inhibition affects pre-mRNA splicing of many genes in the TLR signaling pathway.

<p>The schematic depicts the Toll-like receptor signaling pathway (KEGG map04620) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref143" target="_blank">143</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref144" target="_blank">144</a>] with genes whose splicing is altered (DEXSeq analysis) when SF3a1 is inhibited color coded as follows: red (altered in the absence or presence of LPS), green (only in the absence of LPS), blue (only in the presence of LPS). Additionally, altered splicing of two genes in purple was identified in our other analyses. Generated using DAVID [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref053" target="_blank">53</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref054" target="_blank">54</a>].</p

Pathways affected by LPS Treatment.

<p>*Innate immune signaling pathways underlined.</p><p>Pathways affected by LPS Treatment.</p