iTRAQ Identification of Candidate Serum Biomarkers Associated with Metastatic Progression of Human Prostate Cancer

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer ‘BPH’, (ii) localised cancer with no evidence of progression, ‘non-progressing’ (iii) localised cancer with evidence of biochemical progression, ‘progressing’, and (iv) bone metastasis at presentation ‘metastatic’. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and ‘panels’ of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation

Natural Mutagenesis-Enabled Global Proteomic Study of Metabolic and Carbon Source Implications in Mutant Thermoacidophillic Archaeon <i>Sulfolobus solfataricus</i> PBL2025

The thermoacidophilic crenarchaeon <i>Sulfolobus solfataricus</i> has been widely used as a model organism for archaeal systems biology research. Investigation using its spontaneous mutant PBL2025 provides an effective metabolic baseline to study subsequent mutagenesis-induced functional process shifts as well as changes in feedback inhibitions. Here, an untargeted metabolic investigation using quantitative proteomics and metabolomics was performed to correlate changes in <i>S. solfataricus</i> strains P2 against PBL2025 and under both glucose and tryptone. The study is combined with pathway enrichment analysis to identify prominent proteins with differential stoichiometry. Proteome level quantification reveals that over 20% of the observed overlapping proteome is differentially expressed under these conditions. Metabolic-induced differential expressions are observed along the central carbon metabolism, along with 12 other significantly regulated pathways. Current findings suggest that PBL2025 is able to compensate through the induction of carbon metabolism, as well as other anabolic pathways such as Val, Leu and iso-Leu biosynthesis. Studying protein abundance changes after changes in carbon sources also reveals distinct differences in metabolic strategies employed by both strains, whereby a clear down-regulation of carbohydrate and nucleotide metabolism is observed for P2, while a mixed response through down-regulation of energy formation and up-regulation of glycolysis is observed for PBL2025. This study contributes, to date, the most comprehensive network of changes in carbohydrate and amino acid pathways using the complementary systems biology observations at the protein and metabolite levels. Current findings provide a unique insight into molecular processing changes through natural (spontaneous) metabolic rewiring, as well as a systems biology understanding of the metabolic elasticity of thermoacidophiles to environmental carbon source change, potentially guiding more efficient directed mutagenesis in archaea

HDX-MS study on garadacimab binding to activated FXII reveals potential binding interfaces through differential solvent exposure

ABSTRACTHageman factor (FXII) is an essential component in the intrinsic coagulation cascade and a therapeutic target for the prophylactic treatment of hereditary angioedema (HAE). CSL312 (garadacimab) is a novel high-affinity human antibody capable of blocking activated FXII activity that is currently undergoing Phase 3 clinical trials in HAE. Structural studies using hydrogen/deuterium exchange coupled to mass spectrometry revealed evidence of interaction between the antibody and regions surrounding the S1 specificity pocket of FXII, including the 99-loop, 140-loop, 180-loop, and neighboring regions. We propose complementarity-determining regions (CDRs) in heavy-chain CDR2 and CDR3 as potential paratopes on garadacimab, and the 99-loop, 140-loop, 180-loop, and 220-loop as binding sites on the beta chain of activated FXII (β-FXIIa)

Proteins showing significant differential expression (up-regulated and down-regulated) according to disease progression.

<p>The list of differentially expressed proteins shown are based on comparisons between non-progressing versus BPH; progressing versus non-progressing and metastatic versus progressing groups. Note the differential expression of proteins either individually (black font), as pairs (blue, orange and purple fonts), or as a panel (≥3 proteins, green and red fonts). (*) = identified as a single high confidence peptide.</p

The expression of eEF1A1 and eEF1A2 isoforms in human prostate cancer cell lines.

<p>(A), Western blotting performed on prostate cancer cell lines using an antibody reactive to both the eEF1A1 and eEF1A2 isoforms. GAPDH was used as a loading control. Twenty-five micrograms of protein were loaded in each lane. Lanes 1–11: 1 = LNCaP; 2 = LNCaP-LN3; 3 = LNCaP-Pro5; 4 = LNCaP-C42; 5 = LNCaP-C4-2B; 6 = DuCaP; 7 = VCaP; 8 = Du145; 9 = PC-3; 10 = PC-3M; 11 = PC-3M-LN4. (B), Reverse-transcription PCR specific for the eEF1A1 and eEF1A2 isoforms, performed using mRNA extracted from prostate cancer cell lines: L = LNCaP; P = PC-3; V = VCaP; D = DuCaP. Control (Co), PCR was performed without mRNA. Relative equal expression of eEF1A1 and eEF1A2 mRNA can be seen in all cell lines tested (i.e. relative intensity ratio of 1.0, using Quantity One). (C), Sections of the DNA sequence chromatograms generated by sequencing the PCR products from the LNCaP cell line, confirming the specificity of the PCR primers used. Nucleotide bases unique to each isoform are marked by arrows.</p