Protein Abundance Levels Research Articles

Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering.

Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and we also show that two of the Hmt1 methylation targets can suppress noise. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.

Milk-derived miRNA profiles elucidate molecular pathways that underlie breast dysfunction in women with common genetic variants in SLC30A2

Studies in humans and pre-clinical animal models show milk-derived miRNAs reflect mammary gland function during lactation. The zinc transporter SLC30A2/ZnT2 plays a critical role in mammary gland function; ZnT2-null mice have profound defects in mammary epithelial cell (MEC) polarity and secretion, resulting in sub-optimal lactation. Non-synonymous genetic variation in SLC30A2 is common in humans, and several common ZnT2 variants are associated with changes in milk components that suggest breast dysfunction in women. To identify novel mechanisms through which dysfunction might occur, milk-derived miRNA profiles were characterized in women harboring three common genetic variants in SLC30A2 (D103E, T288S, and Exon 7). Expression of ten miRNAs differed between genotypes, and contributed to distinct spatial separation. Studies in breast milk and cultured MECs confirmed expression of ZnT2 variants alters abundance of protein levels of several predicted mRNA targets critical for breast function (PRLR, VAMP7, and SOX4). Moreover, bioinformatic analysis identified two novel gene networks that may underlie normal MEC function. Thus, we propose that genetic variation in genes critical for normal breast function such as SLC30A2 has important implications for lactation performance in women, and that milk-derived miRNAs can be used to identify novel mechanisms and for diagnostic potential.

N-Glycoproteomic Profiling Reveals Alteration In Extracellular Matrix Organization In Non-Type Bladder Carcinoma.

Treatment of advanced and metastatic bladder carcinoma is often ineffective and displays variable clinical outcomes. Studying this aggressive molecular subtype of bladder carcinoma will lead to better understanding of the pathogenesis which may lead to the identification of new therapeutic strategies. The non-type bladder subtype is phenotypically mesenchymal and has mesenchymal features with a high metastatic ability. Post-translational addition of oligosaccharide residues is an important modification that influences cellular functions and contributes to disease pathology. Here, we report the comparative analysis of N-linked glycosylation across bladder cancer subtypes. To analyze the glycosite-containing peptides, we carried out LC-MS/MS-based quantitative proteomic and glycoproteomic profiling. We identified 1299 unique N-linked glycopeptides corresponding to 460 proteins. Additionally, we identified 118 unique N-linked glycopeptides corresponding to 84 proteins to be differentially glycosylated only in non-type subtypes as compared to luminal/basal subtypes. Most of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 55 differentially expressed glycoproteins showed no significant change at the protein abundance level, representing that the glycosylation site occupancy was changed between the non-type subtype and luminal/basal subtypes. Importantly, the extracellular matrix organization pathway was dysregulated in the non-type subtype of bladder carcinoma. N-glycosylation modifications in the extracellular matrix organization proteins may be a contributing factor for the mesenchymal aggressive phenotype in non-type subtype. These aberrant protein glycosylation would provide additional avenues to employ glycan-based therapies and may lead to the identification of novel therapeutic targets.

A Suppressor Mutation Partially Reverts the xantha Trait via Lowered Methylation in the Promoter of Genomes Uncoupled 4 in Rice.

The xantha trait of a yellow leaf rice mutant (HYB), controlled epigenetically by elevated CHG methylation of the genomes uncoupled 4 (OsGUN4) promoter, has reduced chlorophyll content, altered tetrapyrrole biosynthesis, and deregulated transcription of photosynthesis-associated nuclear genes (PhANGs) compared to its wild-type progenitor Longtefu B (LTB). In the present study, we identified a suppressor mutant (CYB) of HYB and characterized its genetic, molecular, and physiological basis of the mutant phenotype. We found that the light-green phenotype of CYB was caused by a suppressor mutation in an unknown gene other than OsGUN4. Compared to HYB, the CHG methylation in the OsGUN4 promoter was reduced, while OsGUN4 transcript and protein abundance levels were increased in CYB. The contents of total chlorophyll and its intermediate metabolites (except protoporphyrin IX) in CYB plants were intermediate between HYB and LTB. The expression levels of 30 genes involved in tetrapyrrole biosynthesis in CYB were all partially reverted to those of LTB, so were the PhANGs. In summary, a suppressor mutation caused the reversion of the xantha trait via reducing CHG methylation in OsGUN4 promoter.

Multi-omics Characterization of Interaction-mediated Control of Human Protein Abundance levels

Proteogenomic studies of cancer samples have shown that copy-number variation can be attenuated at the protein level for a large fraction of the proteome, likely due to the degradation of unassembled protein complex subunits. Such interaction-mediated control of protein abundance remains poorly characterized. To study this, we compiled genomic, (phospho)proteomic and structural data for hundreds of cancer samples and find that up to 42% of 8,124 analyzed proteins show signs of post-transcriptional control. We find evidence of interaction-dependent control of protein abundance, correlated with interface size, for 516 protein pairs, with some interactions further controlled by phosphorylation. Finally, these findings in cancer were reflected in variation in protein levels in normal tissues. Importantly, expression differences due to natural genetic variation were increasingly buffered from phenotype differences for highly attenuated proteins. Altogether, this study further highlights the importance of posttranscriptional control of protein abundance in cancer and healthy cells.

Synovial fluid proteome changes in ACL injury-induced posttraumatic osteoarthritis: Proteomics analysis of porcine knee synovial fluid.

Surgical transection of the anterior cruciate ligament (ACL) in the porcine model leads to posttraumatic osteoarthritis if left untreated. However, a recently developed surgical treatment, bridge-enhanced ACL repair, prevents further cartilage damage. Since the synovial fluid bathes all the intrinsic structures of knee, we reasoned that a comparative analysis of synovial fluid protein contents could help to better understand the observed chondroprotective effects of the bridge-enhanced ACL repair. We hypothesized that post-surgical changes in the synovial fluid proteome would be different in the untreated and repaired knees, and those changes would correlate with the degree of cartilage damage. Thirty adolescent Yucatan mini-pigs underwent unilateral ACL transection and were randomly assigned to either no further treatment (ACLT, n = 14) or bridge-enhanced ACL repair (BEAR, n = 16). We used an isotopically labeled high resolution LC MS/MS-based proteomics approach to analyze the protein profile of synovial fluid at 6 and 12 months after ACL transection in untreated and repaired porcine knees. A linear mixed effect model was used to compare the normalized protein abundance levels between the groups at each time point. Bivariate linear regression analyses were used to assess the correlations between the macroscopic cartilage damage (total lesion area) and normalized abundance levels of each of the identified secreted proteins. There were no significant differences in cartilage lesion area or quantitative abundance levels of the secreted proteins between the ACLT and BEAR groups at 6 months. However, by 12 months, greater cartilage damage was seen in the ACLT group compared to the BEAR group (p = 0.005). This damage was accompanied by differences in the abundance levels of secreted proteins, with higher levels of Vitamin K-dependent protein C (p = 0.001), and lower levels of Apolipoprotein A4 (p = 0.021) and Cartilage intermediate layer protein 1 (p = 0.049) in the ACLT group compared to the BEAR group. There were also group differences in the secreted proteins that significantly changed in abundance between 6 and 12 months in ACLT and BEAR knees. Increased concentration of Ig lambda-1 chain C regions and decreased concentration of Hemopexin, Clusterin, Coagulation factor 12 and Cartilage intermediate layer protein 1 were associated with greater cartilage lesion area. In general, ACLT knees had higher concentrations of pro-inflammatory proteins and lower concentrations of anti-inflammatory proteins than BEAR group. In addition, the ACLT group had a lower and declining synovial concentrations of CILP, in contrast to a consistently high abundance of CILP in repaired knees. These differences suggest that the knees treated with bridge-enhanced ACL repair may be maintaining an environment that is more protective of the extracellular matrix, a function which is not seen in the ACLT knees.

FSH inhibits AMH to support ovarian estradiol synthesis in infantile mice

Anti-Müllerian hormone (AMH) regulates ovarian function in cyclic females, notably by preventing premature follicle-stimulating hormone (FSH)-mediated follicular growth and steroidogenesis. Its expression in growing follicles is controlled by FSH and by estradiol (E2). In infantile females, there is a transient increase in the activity of the gonadotrope axis, as reflected by elevated levels of both gonadotropins and E2. We previously demonstrated in mice that elevated FSH concentrations are necessary to induce E2 production by preantral/early antral follicles through the stimulation of aromatase expression without supporting their growth. However, whether this action of FSH could involve AMH is unknown. Here, we show that Amh mRNA and protein abundance and serum AMH levels are elevated in infantile mouse females, compared with those in adults. By experimentally manipulating FSH and E2 levels in infantile mice, we demonstrate that high FSH concentrations lower Amh expression specifically in preantral/early antral follicles, whereas E2 has no effect. Importantly, treatment of infantile ovaries in organotypic cultures with AMH decreases FSH-mediated expression of Cyp19a1 aromatase, but it does not alter the expression of cyclin D2-mediating granulosa cell proliferation. Overall, our data indicate that the infantile elevation in FSH levels suppresses Amh expression in preantral/early antral follicles, thereby favoring Cyp19a1 aromatase expression and E2 production. Together with recent discoveries that AMH can act on both the hypothalamus and the pituitary to increase gonadotropin levels, this work suggests that AMH is a critical regulator of the gonadotrope axis during the infantile period, thereby contributing to adult reproductive function programming.

Pacific geoduck (Panopea generosa) resilience to natural pH variation

Pacific geoduck aquaculture is a growing industry, however, little is known about how geoduck respond to varying environmental conditions, or how the industry will fare under projected climate conditions. To understand how geoduck production may be impacted by low pH associated with ocean acidification, multi-faceted environmental heterogeneity needs to be included to understand species and community responses. In this study, eelgrass habitats and environmental heterogeneity across four estuarine bays were leveraged to examine low pH effects on geoduck under different natural regimes, using targeted proteomics to assess physiology. Juvenile geoduck were deployed in eelgrass and adjacent unvegetated habitats for 30 days while pH, temperature, dissolved oxygen, and salinity were monitored. Across the four bays, pH was lower in unvegetated habitats compared to eelgrass habitats. However this did not impact geoduck growth, survival, or proteomic abundance patterns in gill tissue. Temperature and dissolved oxygen differences across all locations corresponded to differences in growth and targeted protein abundance patterns. Specifically, three protein abundance levels (trifunctional-enzyme β-subunit, puromycin-sensitive aminopeptidase, and heat shock protein 90-α) and shell growth positively correlated with dissolved oxygen variability and inversely correlated with mean temperature. These results demonstrate that geoduck may be resilient to low pH in a natural setting, but other abiotic factors (i.e. temperature, dissolved oxygen variability) may have a greater influence on geoduck physiology. In addition this study contributes to the understanding of how eelgrass patches influences water chemistry.

The anti-apoptotic effect of nerve growth factor on propofol-induced neurotoxicity in hippocampal neurons is Rac1 dependent.

Propofol has been considered as a near-ideal anesthetic agent since its introduction 40 years ago. However, the side effects of propofol including bacterial contamination, hyperlipidemia, and neurotoxicity also aroused attention. Nerve growth factor (NGF) plays a pivotal role in the development, differentiation, and survival of the neurons of the peripheral and central nervous system. In the present study, we found that NGF alleviated the apoptosis induced by propofol in hippocampal neurons. Furthermore, NGF treatment augmented the protein abundance and mRNA level of Rac1 while silencing Rac1 significantly blunted the effects of NGF upon propofol-induced apoptosis. In conclusion, NGF decreased propofol-induced apoptosis and this effect was Rac1 dependent.

Proteomic Signatures of Clostridium difficile Stressed with Metronidazole, Vancomycin, or Fidaxomicin.

The anaerobic pathogen Clostridium difficile is of growing significance for the health care system due to its increasing incidence and mortality. As C. difficile infection is both supported and treated by antibiotics, a deeper knowledge on how antimicrobial agents affect the physiology of this important pathogen may help to understand and prevent the development and spreading of antibiotic resistant strains. As the proteomic response of a cell to stress aims at counteracting the harmful effects of this stress, it can be expected that the pattern of a pathogen’s responses to antibiotic treatment will be dependent on the antibiotic mechanism of action. Hence, every antibiotic treatment is expected to result in a specific proteomic signature characterizing its mode of action. In the study presented here, the proteomic response of C. difficile 630∆erm to vancomycin, metronidazole, and fidaxomicin stress was investigated on the level of protein abundance and protein synthesis based on 2D PAGE. The quantification of 425 proteins of C. difficile allowed the deduction of proteomic signatures specific for each drug treatment. Indeed, these proteomic signatures indicate very specific cellular responses to each antibiotic with only little overlap of the responses. Whereas signature proteins for vancomycin stress fulfil various cellular functions, the proteomic signature of metronidazole stress is characterized by alterations of proteins involved in protein biosynthesis and protein degradation as well as in DNA replication, recombination, and repair. In contrast, proteins differentially expressed after fidaxomicin treatment can be assigned to amino acid biosynthesis, transcription, cell motility, and the cell envelope functions. Notably, the data provided by this study hint also at so far unknown antibiotic detoxification mechanisms.

Differences in sperm protein abundance and carbonylation level in bull ejaculates of low and high quality.

In breeding and insemination centres, significant variation in bull ejaculate quality is often observed between individuals and also within the same individual. Low-quality semen does not qualify for cryopreservation and is rejected, generating economic loss. The mechanisms underlying the formation of low-quality ejaculates are poorly understood; therefore, the aim of the present study was to investigate the proteomic differences and oxidative modifications (measured as changes in protein carbonylation level) of bull ejaculates of low and high quality. Flow cytometry and computer-assisted sperm analysis were used to assess differences in viability, reactive oxygen species (ROS) level, and sperm motility. To analyse changes in protein abundance, two-dimensional difference gel electrophoresis (2D-DIGE) was performed. Western blotting in conjunction with two-dimensional electrophoresis (2D-oxyblot) was used to quantitate carbonylated sperm proteins. Proteins were identified using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight spectrometry. High quality ejaculates were characterised by higher sperm motility, viability, concentration, and a lower number of ROS-positive cells (ROS+). We found significant differences in the protein profile between high- and low-quality ejaculates, and identified 14 protein spots corresponding to 10 proteins with differences in abundance. The identified sperm proteins were mainly associated with energetic metabolism, capacitation, fertilisation, motility, and cellular detoxification. High-quality ejaculates were characterised by a high abundance of extracellular sperm surface proteins, likely due to more efficient secretion from accessory sex glands and/or epididymis, and a low abundance of intracellular proteins. Our results show that sperm proteins in low-quality ejaculates are characterised by a high carbonylation level. Moreover, we identified, for the first time, 14 protein spots corresponding to 12 proteins with differences in carbonylation level between low- and high-quality ejaculates. The carbonylated proteins were localised mainly in mitochondria or their immediate surroundings. Oxidative damage to proteins in low-quality semen may be associated with phosphorylation/dephosphorylation disturbances, mitochondrial dysfunction, and motility apparatus disorders. Our results contribute to research regarding the mechanism by which low- and high-quality ejaculates are formed and to the identification of sperm proteins that are particularly sensitive to oxidative damage.

High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect.

Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3, a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development.

Comparative Proteomic Analysis of Two Ralstonia solanacearum Isolates Differing in Aggressiveness

Ralstonia solanacearum is a soil-borne, plant xylem-infecting pathogen that causes the devastating bacterial wilt (BW) disease in a number of plant species. In the present study, two R. solanacearum strains with different degrees of aggressiveness―namely RsH (pathogenic to Hawaii 7996, a tomato cultivar resistant against most strains) and RsM (non-pathogenic to Hawaii 7996) were identified. Phylogenetic analysis revealed that both RsM and RsH belonged to phylotype I. To further elucidate the underlying mechanism of the different pathotypes between the two strains, we performed a comparative proteomics study on RsM and RsH in rich and minimal media to identify the change in the level of protein abundance. In total, 24 differential proteins were identified, with four clusters in terms of protein abundance. Further bioinformatics exploration allowed us to classify these proteins into five functional groups. Notably, the pathogenesis of RsM and RsH was particularly characterized by a pronounced difference in the abundance of virulence- and metabolism-related proteins, such as UDP-N-acetylglucosamine 2-epimerase (epsC) and isocitrate lyase (ICL), which were more abundant in the high pathogenicity strain RsH. Thus, we propose that the differences in pathogenicity between RsM and RsH can possibly be partially explained by differences in extracellular polysaccharide (EPS) and glyoxylate metabolism-related proteins.

Abstract 2708: Imputation-free analysis of high throughput TMT proteomics of 116 lung squamous samples

Abstract Introduction: Chemical labeling of peptides using tandem mass tags (TMT) is a “barcoding” strategy, enabling relative protein quantification across a single panel of samples (as opposed to each run separately). Each multiplex assay is, effectively, its own "batch" of samples, and thus direct comparison of intensities between TMT multiplexes is problematic. Additionally, although there is relatively little missing data within a single plex, there can be large differences in missingness across plexes, with the two types of missingness exhibiting different behavior (infrequent and biased towards low abundances within-plex; more frequent and more stochastic between-plex). We have addressed these issues by developing new pipelines for data normalization, protein-level rollup, and downstream clustering, which seek to minimize the negative impact of missingness. This method development was driven by, and applied to, a set of 116 human lung squamous (SQLC) tumors, with the aim of improving the strength of down-stream biological signal and interpretation. Experiment: TMT analysis was performed on 116 SQLC samples. Each 6-plex contained 4 tumors and 2 pool replicates. The shared pool of 116 tumors was assayed on every multiplex to allow for controlling for variability between plexes, with one pool in ch-126 and the other varying channel between plexes. IDPicker was used for spectral quantification. Spectra abundances were normalized within-plex, and ratios calculated for each channel against the ch-126 pool. Spectra-level ratios were rolled up into protein-level ratios using the geometric mean of ratios within each protein group. Geometric mean protein-level abundance rollup was performed on abundances for each ch-126 pool, normalized across pools, and the geometric mean calculated for each protein group across pools. These mean protein-level abundances were then used to scale the ratios back into final normalized abundances. Average linkage hierarchical clustering was performed on abundance z-scores using a novel distance metric, calculated as the root mean squared deviation (RMSD) of points present in both vectors, divided by a binary presence/absence similarity coefficient such as Ochiai similarity. Results: After normalization, principal component analysis showed no batch effect due to differences between plexes. Heat maps generated using the novel distance metric exhibited improved biological signal over RMSD alone. Tumors cluster into 3 major groupings: high immune + low transcriptional/translational activity, low immune + high transcriptional/translational activity, and samples with medium levels of both. Conclusion: Missingness-aware methods of shared-pool TMT normalization and clustering minimize the negative impact of missingness and yield strong biological signal. Preliminary results suggest that immune response is a major source of differences between lung squamous tumors. Citation Format: Eric A. Welsh, Paul A. Stewart, Matthew C. Chambers, Guolin Zhang, Bin Fang, Steven A. Eschrich, John M. Koomen, Eric B. Haura. Imputation-free analysis of high throughput TMT proteomics of 116 lung squamous samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2708.

Ribosome elongating footprints denoised by wavelet transform comprehensively characterize dynamic cellular translation events.

Translation is dynamically regulated during cell development and stress response. In order to detect actively translated open reading frames (ORFs) and dynamic cellular translation events, we have developed a computational method, RiboWave, to process ribosome profiling data. RiboWave utilizes wavelet transform to denoise the original signal by extracting 3-nt periodicity of ribosomes and precisely locate their footprint denoted as Periodic Footprint P-site (PF P-site). Such high-resolution footprint is found to capture the full track of actively elongating ribosomes, from which translational landscape can be explicitly characterized. We compare RiboWave with several published methods, like RiboTaper, ORFscore and RibORF, and found that RiboWave outperforms them in both accuracy and usage when defining actively translated ORFs. Moreover, we show that PF P-site derived by RiboWave shows superior performance in characterizing the dynamics and complexity of cellular translatome by accurately estimating the abundance of protein levels, assessing differential translation and identifying dynamic translation frameshift.

Proteo-metabolomics reveals compensation between ischemic and non-injured contralateral kidneys after reperfusion

Ischaemia and reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI), which contributes to high morbidity and mortality rates in a wide range of injuries as well as the development of chronic kidney disease. The cellular and molecular responses of the kidney to IRI are complex and not fully understood. Here, we used an integrated proteomic and metabolomic approach to investigate the effects of IRI on protein abundance and metabolite levels. Rat kidneys were subjected to 45 min of warm ischaemia followed by 4 h and 24 h reperfusion, with contralateral and separate healthy kidneys serving as controls. Kidney tissue proteomics after IRI revealed elevated proteins belonging to the acute phase response, coagulation and complement pathways, and fatty acid (FA) signalling. Metabolic changes were already evident after 4 h reperfusion and showed increased level of glycolysis, lipids and FAs, whilst mitochondrial function and ATP production was impaired after 24 h. This deficit was partially compensated for by the contralateral kidney. Such a metabolic balance counteracts for the developing energy deficit due to reduced mitochondrial function in the injured kidney.

NF-κB-mediated metabolic remodelling in the inflamed heart in acute viral myocarditis

Acute viral myocarditis (VM), characterised by leukocyte infiltration and dysfunction of the heart, is an important cause of sudden cardiac death in young adults. Unfortunately, to date, the pathological mechanisms underlying cardiac failure in VM remain incompletely understood. In the current study, we investigated if acute VM leads to cardiac metabolic rewiring and if this process is driven by local inflammation. Transcriptomic analysis of cardiac biopsies from myocarditis patients and a mouse model of VM revealed prominent reductions in the expression of a multitude of genes involved in mitochondrial oxidative energy metabolism. In mice, this coincided with reductions in high-energy phosphate and NAD levels, as determined by Imaging Mass Spectrometry, as well as marked decreases in the activity, protein abundance and mRNA levels of various enzymes and key regulators of cardiac oxidative metabolism. Indicative of fulminant cardiac inflammation, NF-κB signalling and inflammatory cytokine expression were potently induced in the heart during human and mouse VM. In cultured cardiomyocytes, cytokine-mediated NF-κB activation impaired cardiomyocyte oxidative gene expression, likely by interfering with the PGC-1 (peroxisome proliferator-activated receptor (PPAR)-γ co-activator) signalling network, the key regulatory pathway controlling cardiomyocyte oxidative metabolism. In conclusion, we provide evidence that acute VM is associated with extensive cardiac metabolic remodelling and our data support a mechanism whereby cytokines secreted primarily from infiltrating leukocytes activate NF-κB signalling in cardiomyocytes thereby inhibiting the transcriptional activity of the PGC-1 network and consequently modulating myocardial energy metabolism.

Unification of Protein Abundance Datasets Yields a Quantitative Saccharomyces cerevisiae Proteome

Protein activity is the ultimate arbiter of function in most cellular pathways, and protein concentration is fundamentally connected to protein action. While the proteome of yeast has been subjected to themost comprehensive analysis of any eukaryote, existing datasets are difficult to compare, and thereis no consensus abundance value for each protein.We evaluated 21 quantitative analyses of the S.cerevisiae proteome, normalizing and converting all measurements of protein abundance into the intuitive measurement of absolute molecules per cell. We estimate the cellular abundance of 92% of the proteins in the yeast proteome and assess the variation in each abundance measurement. Using our protein abundance dataset, we find that a global response to diverse environmental stresses is not detected at the level of protein abundance, we find that protein tags have only a modest effect on protein abundance, and we identify proteins that are differentially regulated at the mRNA abundance, mRNA translation, and protein abundance levels.

Refining Transcriptome Gene Catalogs by MS-Validation of Expressed Proteins.

Protein sequence identification by tandem mass spectroscopy (LC-MS/MS) identifies thousands of protein sequences even in complex mixtures, and provides valuable insight into the biological functions of different cells. For non-model organisms, transcriptomes are generally used to allow peptide identification, an important addition to their use as a gene catalog allowing the potential metabolic activities of cells to be determined. We used LC-MS/MS data to identify which of the six possible reading frames in the transcriptome was actually used by the cell to make protein, and asked whether this would have an impact on downstream analyses using the dataset. We combined results from several LC-MS/MS experiments designed to identify peptide sequences in extracts from the dinoflagellate Lingulodinium polyedra using a 74 655-sequence transcriptome. We compiled a list of 6628 translated nucleic acid sequences that contained the ensemble of peptide matches (termed MS-validated sequences) and assessed the similarity in downstream analyses between this data set and the 6628 nucleic acid sequences from which they were derived. When compared with BLASTx analyses of the DNA sequences, the MS-validated protein-sequences-analyzed using BLASTp showed differences in gene ontology, had more identified BLAST hits, and contained more KEGG pathway enzymes. The MS-validated protein sequences also differ from datasets containing longest open reading frame (ORF) protein sequences. We also note a poor correlation between the levels of protein and mRNA abundance, a comparison not previously performed for dinoflagellates. The differences observed between analyses of MS-validated protein sequence and nucleic acid sequence datasets suggest use of the former may provide a more accurate representation of cellular capacity than the latter. Developing MS-validated protein sequence datasets may also speed interpretation of MS-MS spectra in bottom up proteomics experiments.

Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera

BackgroundRapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology.ResultsComparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects.ConclusionsOur results identify a burst of genetic novelty amongst sperm proteins that may be associated with the origin of heteromorphic spermatogenesis in ancestral Lepidoptera and/or the subsequent evolution of this system. This pattern of genomic diversification is distinct from the remainder of the genome and thus suggests that this transition has had a marked impact on lepidopteran genome evolution. The identification of abundant sperm proteins unique to Lepidoptera, including proteins distinct between specific lineages, will accelerate future functional studies aiming to understand the developmental origin of dichotomous spermatogenesis and the functional diversification of the fertilization incompetent apyrene sperm morph.