Effect of pH on the denitrification proteome of the soil bacterium Paracoccus denitrificans PD1222

Denitrification is a respiratory process by which nitrate is reduced to dinitrogen. Incomplete denitrification results in the emission of the greenhouse gas nitrous oxide and this is potentiated in acidic soils, which display reduced denitrification rates and high N2O/N2 ratios compared to alkaline soils. In this work, impact of pH on the proteome of the soil denitrifying bacterium Paracoccus denitrificans PD1222 was analysed with nitrate as sole energy and nitrogen source under anaerobic conditions at pH ranging from 6.5 to 7.5. Quantitative proteomic analysis revealed that the highest difference in protein representation was observed when the proteome at pH 6.5 was compared to the reference proteome at pH 7.2. However, this difference in the extracellular pH was not enough to produce modification of intracellular pH, which was maintained at 6.5 ± 0.1. The biosynthetic pathways of several cofactors relevant for denitrification and nitrogen assimilation like cobalamin, riboflavin, molybdopterin and nicotinamide were negatively affected at pH 6.5. In addition, peptide representation of reductases involved in nitrate assimilation and denitrification were reduced at pH 6.5. Data highlight the strong negative impact of pH on NosZ synthesis and intracellular copper content, thus impairing active NosZ assembly and, in turn, leading to elevated nitrous oxide emissions.

pCRM1exportome: database of predicted CRM1-dependent Nuclear Export Signal (NES) motifs in cancer-related genes

Motivation The consensus pattern of Nuclear Export Signal (NES) is a short sequence motif that is commonly identified in protein sequences, whether the motif acts as an NES (true positive) or not (false positive). Finding more plausible NES functioning regions among the vast array of consensus-matching segments would provide an interesting resource for further experimental validation. Better defined NES should also allow meaningful mapping of cancer-related mutation positions, leading to plausible explanations for the relationship between nuclear export and disease. Results Possible NES candidate regions are extracted from the cancer-related human reference proteome. Extracted NES are scored for reliability by combining sequence-based and structure-based approaches. The confidently identified NES candidate motifs were checked for overlap with cancer-related mutation positions annotated in the COSMIC database. Among the ∼700 cancer-related sequences in the COSMIC Cancer Gene Census, 178 sequences are predicted to have possible NES motifs containing cancer-related mutations at their key positions. These lists are organized into our database (pCRM1exportome), and other protein sequences in the human reference proteome can also be retrieved by their UniProt IDs. Availability and implementation The database is freely available at http://prodata.swmed.edu/pCRM1exportome. Supplementary information Supplementary data are available at Bioinformatics online.

Integrative analysis of proteome and transcriptome dynamics duringBacillus subtilisspore revival

Bacillus subtilisforms highly resistant, metabolically inactive dormant spores upon nutrient limitation. These endospores pose challenges to the food and medical sectors. Spores reactivate their metabolism upon contact with germinants and develop into vegetative cells. The activation of the molecular machinery that triggers the progress of germination and spore outgrowth is still unsettled. To gain further insight in spore germination and outgrowth processes, the transcriptome and proteome changeover during spore germination and outgrowth to vegetative cells, was analysed.B. subtilistranscriptome analysis allow us to trace the different functional groups of genes expressed. For each time-point sample, the change in the spore proteome was quantitatively monitored relative to the reference proteome of15N metabolically labelled vegetative cells. We observed until the phase transition, i.e. completion of germination, no significant change in the proteome. We have identified 36 transcripts present abundantly in the dormant spores. This number is in close agreement with the previous findings. These transcripts mainly belong to the genes encoding small acid soluble proteins (sspE, sspO, sspI, sspK, sspF) and proteins with uncharacterized functions. We observed in total 3152 differentially expressed genes, but ‘only’ 323 differentially expressed proteins (total 451 proteins identified and quantified). Our data shows that 173 proteins from dormant spores, both spore unique proteins and protein shared with vegetative cells, are lost during the phase transitioning period. This loss is in addition to the active protein degradation, undertaken by the spore proteases such as Gpr, as germination and outgrowth proceeds. Further analysis is required to functionally interpret the observed protein loss. The observed diverse timing of the synthesis of different protein sets reveals a putative core-strategy of the revival of ‘life’ starting from theB. subtilisspore.

BEWARE OF MOVING TARGETS: REFERENCE PROTEOME CONTENT FLUCTUATES SUBSTANTIALLY OVER THE YEARS

Reference proteomes are generated by increasingly sophisticated annotation pipelines as part of regular genome build releases; yet, the corresponding changes in reference proteomes' content are dramatic. In the history of the NCBI-curated human proteome, the total number of entries has remained roughly constant but approximately half of the proteins from the 2003 build 33 are no longer represented by entries in current releases, while about the same number of new proteins have been added (for sequence identity thresholds 50–90%). Although mostly hypothetical proteins are affected, there are also spectacular cases of entry removal/addition of well studied proteins. The changes between the 2003 and recent human proteomes are in a similar order of magnitude as the differences between recent human and chimpanzee proteome releases. As an application example, we show that the proteome fluctuations affect the interpretation (about 74% of hits) of organelle-specific mass-spectrometry data. Although proteome quality tends to improve with more recent releases as, for example, the fraction of proteins with functional annotation has increased over time, existing evidence implies that, apparently, the proteome content still remains incomplete, not just pertaining to isoforms/sequence variants but also to proteins and their families that are clearly distinct.