Control of cell state transitions.

Understanding cell state transitions and purposefully controlling them is a longstanding challenge in biology. Here we present cell state transition assessment and regulation (cSTAR), an approach for mapping cell states, modelling transitions between them and predicting targeted interventions to convert cell fate decisions. cSTAR uses omics data as input, classifies cell states, and develops a workflow that transforms the input data into mechanistic models that identify a core signalling network, which controls cell fate transitions by influencing whole-cell networks. By integrating signalling and phenotypic data, cSTAR models how cells manoeuvre in Waddington's landscape1 and make decisions about which cell fate to adopt. Notably, cSTAR devises interventions to control the movement of cells in Waddington's landscape. Testing cSTAR in a cellular model of differentiation and proliferation shows a high correlation between quantitative predictions and experimental data. Applying cSTAR to different types of perturbation and omics datasets, including single-cell data, demonstrates its flexibility and scalability and provides new biological insights. The ability of cSTAR to identify targeted perturbations that interconvert cell fates will enable designer approaches for manipulating cellular development pathways and mechanistically underpinned therapeutic interventions.

The H3K36me2 Methyltransferase Nsd1 Demarcates PRC2-Mediated H3K27me2 and H3K27me3 Domains in Embryonic Stem Cells.

The Polycomb repressor complex 2 (PRC2) is composed of the core subunits Ezh1/2, Suz12, and Eed, and it mediates all di- and tri-methylation of histone H3 at lysine 27 in higher eukaryotes. However, little is known about how the catalytic activity of PRC2 is regulated to demarcate H3K27me2 and H3K27me3 domains across the genome. To address this, we mapped the endogenous interactomes of Ezh2 and Suz12 in embryonic stem cells (ESCs), and we combined this with a functional screen for H3K27 methylation marks. We found that Nsd1-mediated H3K36me2 co-locates with H3K27me2, and its loss leads to genome-wide expansion of H3K27me3. These increases in H3K27me3 occurred at PRC2/PRC1 target genes and as de novo accumulation within what were previously broad H3K27me2 domains. Our data support a model in which Nsd1 is a key modulator of PRC2 function required for regulating the demarcation of genome-wide H3K27me2 and H3K27me3 domains in ESCs.

A Family of Vertebrate-Specific Polycombs Encoded by the LCOR/LCORL Genes Balance PRC2 Subtype Activities.

The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, "PRC2 associated LCOR isoform 1" (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.

Differential expression of haptoglobin in individuals at clinical high risk of psychosis and its association with global functioning and clinical symptoms.

BACKGROUND: Immune dysregulation has been observed in patients with schizophrenia or first-episode psychosis, but few have examined dysregulation in those at clinical high-risk (CHR) for psychosis. The aim of this study was to examine whether the peripheral blood-based proteome was dysregulated in those with CHR. Secondly, we examined whether baseline dysregulation was related to current and future functioning and clinical symptoms. METHODS: We used data from participants of the North American Prodromal Longitudinal Studies (NAPLS) 2 and 3 (n = 715) who provided blood samples (Unaffected Comparison subjects (UC) n = 223 and CHR n = 483). Baseline proteomic data was quantified from plasma samples using mass spectrometry. Differential expression was examined between CHR and UC using logistic regression. Psychosocial functioning was measured using the Global Assessment of Functioning scale (GAF). Symptoms were measured using the subscale scores from the Scale of Psychosis-risk Symptoms; positive, negative, general, and disorganised. Three measures of each outcome were included: baseline, longest available follow-up (last follow-up) and most severe follow-up (MSF). Associations between the proteomic data, GAF and symptoms were assessed using ordinal regression. RESULTS: Of the 99 proteins quantified, six were differentially expressed between UC and CHR. However, only haptoglobin (HP) survived FDR-correction (OR:1.45, 95 %CI:1.23-1.69, padj = <0.001). HP was cross-sectionally and longitudinally associated with functioning and symptoms such that higher HP values were associated with poorer functioning and more severe symptoms. Results were evident after stringent adjustment and poorer functioning was observed in both NAPLS cohort separately. CONCLUSION: We demonstrate that elevated HP is robustly observed in those at CHR for psychosis, irrespective of transition to psychosis. HP is longitudinally associated with poorer functioning and greater symptom severity. These results agree with previous reports of increased HP gene expression in individuals at-risk for psychosis and with the dysfunction of the acute phase inflammatory response seen in psychotic disorders.

Plasma complement and coagulation proteins as prognostic factors of negative symptoms: An analysis of the NAPLS 2 and 3 studies.

INTRODUCTION: Negative symptoms impact the quality of life of individuals with psychosis and current treatment options for negative symptoms have limited effectiveness. Previous studies have demonstrated that complement and coagulation pathway protein levels are related to later psychotic experiences, psychotic disorder, and functioning. However, the prognostic relationship between complement and coagulation proteins and negative symptoms is poorly characterised. METHODS: In the North American Prodrome Longitudinal Studies 2 and 3, negative symptoms in 431 individuals at clinical high-risk for psychosis (mean age: 18.2, SD 3.6; 42.5 % female) were measured at multiple visits over 2 years using the Scale of Psychosis-Risk Symptoms. Plasma proteins were quantified at baseline using mass spectrometry. Four factors were derived to represent levels of proteins involved in the activation or regulation of the complement or coagulation systems. The relationships between standardised protein group factors and serial measurements of negative symptoms over time were modelled using generalised least squares regression. Analyses were adjusted for baseline candidate prognostic factors: negative symptoms, positive symptoms, functioning, depressive symptoms, suicidal ideation, cannabis use, tobacco use, antipsychotic use, antidepressant use, age, and sex. RESULTS: Clinical and demographic prognostic factors of follow-up negative symptoms included negative, positive, and depressive symptoms, functioning, and age. Adjusting for all candidate prognostic factors, the complement regulators group and the coagulation regulators group were identified as prognostic factors of follow-up negative symptoms (ß: 0.501, 95 % CI: 0.160, 0.842; ß: 0.430, 95 % CI: 0.080, 0.780 respectively. The relationship between complement regulator levels and negative symptoms was also observed in NAPLS2 alone (ß: 0.501, 95 % CI: -0.037, 1.039) and NAPLS3 alone, additionally adjusting for BMI (ß: 0.442, 95 % CI: 0.127, 0.757). CONCLUSION: The results indicate that plasma complement and coagulation regulator levels are prognostic factors of negative symptoms, independent of clinical and demographic prognostic factors. These results suggest complement and coagulation regulator levels could have potential utility in informing treatment decisions for negative symptoms in individuals at risk.

Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells.

Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.

Machine learning based prediction and the influence of complement - Coagulation pathway proteins on clinical outcome: Results from the NEURAPRO trial.

BACKGROUND: Functional outcomes are important measures in the overall clinical course of psychosis and individuals at clinical high-risk (CHR), however, prediction of functional outcome remains difficult based on clinical information alone. In the first part of this study, we evaluated whether a combination of biological and clinical variables could predict future functional outcome in CHR individuals. The complement and coagulation pathways have previously been identified as being of relevance to the pathophysiology of psychosis and have been found to contribute to the prediction of clinical outcome in CHR participants. Hence, in the second part we extended the analysis to evaluate specifically the relationship of complement and coagulation proteins with psychotic symptoms and functional outcome in CHR. MATERIALS AND METHODS: We carried out plasma proteomics and measured plasma cytokine levels, and erythrocyte membrane fatty acid levels in a sub-sample (n = 158) from the NEURAPRO clinical trial at baseline and 6 months follow up. Functional outcome was measured using Social and Occupational Functional assessment Score (SOFAS) scale. Firstly, we used support vector machine learning techniques to develop predictive models for functional outcome at 12 months. Secondly, we developed linear regression models to understand the association between 6-month follow-up levels of complement and coagulation proteins with 6-month follow-up measures of positive symptoms summary (PSS) scores and functional outcome. RESULTS AND CONCLUSION: A prediction model based on clinical and biological data including the plasma proteome, erythrocyte fatty acids and cytokines, poorly predicted functional outcome at 12 months follow-up in CHR participants. In linear regression models, four complement and coagulation proteins (coagulation protein X, Complement C1r subcomponent like protein, Complement C4A & Complement C5) indicated a significant association with functional outcome; and two proteins (coagulation factor IX and complement C5) positively associated with the PSS score. Our study does not provide support for the utility of cytokines, proteomic or fatty acid data for prediction of functional outcomes in individuals at high-risk for psychosis. However, the association of complement protein levels with clinical outcome suggests a role for the complement system and the activity of its related pathway in the functional impairment and positive symptom severity of CHR patients.

Complement pathway changes at age 12 are associated with psychotic experiences at age 18 in a longitudinal population-based study: evidence for a role of stress.

The complement cascade is a major component of the immune defence against infection, and there is increasing evidence for a role of dysregulated complement in major psychiatric disorders. We undertook a directed proteomic analysis of the complement signalling pathway (n = 29 proteins) using data-independent acquisition. Participants were recruited from the UK avon longitudinal study of parents and children (ALSPAC) cohort who participated in psychiatric assessment interviews at ages 12 and 18. Protein expression levels at age 12 among individuals who reported psychotic experiences (PEs) at age 18 (n = 64) were compared with age-matched controls (n = 67). Six out of the 29 targeted complement proteins or protein subcomponents were significantly upregulated following correction for multiple comparisons (VTN↑, C1RL↑, C8B↑, C8A↑, CFH↑, and C5↑). We then undertook an unbiased plasma proteomic analysis of mice exposed to chronic social stress and observed dysregulation of 11 complement proteins, including three that were altered in the same direction in individuals with PE (C1R↑, CFH↑, and C5↑). Our findings indicate that dysregulation of the complement protein pathway in blood is associated with incidence of psychotic experiences and that these changes may reflect exposure to stress.

Reconstruction and analysis of a large-scale binary Ras-effector signaling network.

BACKGROUND: Ras is a key cellular signaling hub that controls numerous cell fates via multiple downstream effector pathways. While pathways downstream of effectors such as Raf, PI3K and RalGDS are extensively described in the literature, how other effectors signal downstream of Ras is often still enigmatic. METHODS: A comprehensive and unbiased Ras-effector network was reconstructed downstream of 43 effector proteins (converging onto 12 effector classes) using public pathway and protein-protein interaction (PPI) databases. The output is an oriented graph of pairwise interactions defining a 3-layer signaling network downstream of Ras. The 2290 proteins comprising the network were studied for their implication in signaling crosstalk and feedbacks, their subcellular localizations, and their cellular functions. RESULTS: The final Ras-effector network consists of 2290 proteins that are connected via 19,080 binary PPIs, increasingly distributed across the downstream layers, with 441 PPIs in layer 1, 1660 in layer 2, and 16,979 in layer 3. We identified a high level of crosstalk among proteins of the 12 effector classes. A class-specific Ras sub-network was generated in CellDesigner (.xml file) and a functional enrichment analysis thereof shows that 58% of the processes have previously been associated to a respective effector pathway, with the remaining providing insights into novel and unexplored functions of specific effector pathways. CONCLUSIONS: Our large-scale and cell general Ras-effector network is a crucial steppingstone towards defining the network boundaries. It constitutes a 'reference interactome' and can be contextualized for specific conditions, e.g. different cell types or biopsy material obtained from cancer patients. Further, it can serve as a basis for elucidating systems properties, such as input-output relationships, crosstalk, and pathway redundancy. Video Abstract.

Fam60a defines a variant Sin3a-Hdac complex in embryonic stem cells required for self-renewal.

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a-Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3-positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1-phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.

SETD1A Methyltransferase Is Physically and Functionally Linked to the DNA Damage Repair Protein RAD18.

SETD1A is a SET domain-containing methyltransferase involved in epigenetic regulation of transcription. It is the main catalytic component of a multiprotein complex that methylates lysine 4 of histone H3, a histone mark associated with gene activation. In humans, six related protein complexes with partly nonredundant cellular functions share several protein subunits but are distinguished by unique catalytic SET-domain proteins. We surveyed physical interactions of the SETD1A-complex using endogenous immunoprecipitation followed by label-free quantitative proteomics on three subunits: SETD1A, RBBP5, and ASH2L. Surprisingly, SETD1A, but not RBBP5 or ASH2L, was found to interact with the DNA damage repair protein RAD18. Reciprocal RAD18 immunoprecipitation experiments confirmed the interaction with SETD1A, whereas size exclusion and protein network analysis suggested an interaction independent of the main SETD1A complex. We found evidence of SETD1A and RAD18 influence on mutual gene expression levels. Further, knockdown of the genes individually showed a DNA damage repair phenotype, whereas simultaneous knockdown resulted in an epistatic effect. This adds to a growing body of work linking epigenetic enzymes to processes involved in genome stability.

Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Cell differentiation is directed by extracellular cues and intrinsic epigenetic modifications, which control chromatin organization and transcriptional activation. Central to this process is PRC2, which modulates the di- and trimethylation of lysine 27 on histone 3; however, little is known concerning the direction of PRC2 to specific loci. Here, we have investigated the physical interactome of EZH2, the enzymatic core of PRC2, during retinoic acid-mediated differentiation of neuroepithelial, pluripotent NT2 cells and the dedifferentiation of neuroretinal epithelial ARPE19 cells in response to TGF-ß. We identified Smad3 as an EZH2 interactor in both contexts. Co-occupation of the CDH1 promoter by Smad3 and EZH2 and the cooperative, functional nature of the interaction were established. We propose that the interaction between Smad3 and EZH2 targets the core polycomb assembly to defined regions of the genome to regulate transcriptional repression and forms a molecular switch that controls promoter access through epigenetic mechanisms leading to gene silencing.-Andrews, D., Oliviero, G., De Chiara, L., Watson, A., Rochford, E., Wynne, K., Kennedy, C., Clerkin, S., Doyle, B., Godson, C., Connell, P., O'Brien, C., Cagney, G., Crean, J. Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Triphenyl phosphate is a selective PPARγ modulator that does not induce brite adipogenesis in vitro and in vivo.

Triphenyl phosphate (TPhP) is an environmental PPARγ ligand, and growing evidence suggests that it is a metabolic disruptor. We have shown previously that the structurally similar ligand, tributyltin, does not induce brite adipocyte gene expression. Here, using in vivo and in vitro models, we tested the hypothesis that TPhP is a selective PPARγ ligand, which fails to induce brite adipogenesis. C57BL/6 J male mice were fed either a low or very high-fat diet for 13 weeks. From weeks 7-13, mice were injected intraperitoneally, daily, with vehicle, rosiglitazone (Rosi), or TPhP (10 mg/kg). Compared to Rosi, TPhP did not induce expression of browning-related genes (e.g. Elovl3, Cidea, Acaa2, CoxIV) in mature adipocytes isolated from inguinal adipose. To determine if this resulted from an effect directly on the adipocytes, 3T3-L1 cells and primary human preadipocytes were differentiated into adipocytes in the presence of Rosi or TPhP. Rosi, but not TPhP, induced expression of brite adipocyte genes, mitochondrial biogenesis and cellular respiration. Further, Rosi and TPhP-induced distinct proteomes and phosphoproteomes; Rosi enriched more regulatory pathways related to fatty acid oxidation and mitochondrial proteins. We assessed the role of phosphorylation of PPARγ in these differences in 3T3-L1 cells. Only Rosi protected PPARγ from phosphorylation at Ser273. TPhP gained the ability to stimulate brite adipocyte gene expression in the presence of the CDK5 inhibitor and in 3T3-L1 cells expressing alanine at position 273. We conclude that TPhP is a selective PPARγ modulator that fails to protect PPARγ from phosphorylation at ser273.

Proteomic Analysis Reveals a Strong Association of ß-Catenin With Cadherin Adherens Junctions in Resting Human Platelets.

It was previously demonstrated that the WNT/ß-catenin pathway is present and active in platelets and established that the canonical WNT ligand, WNT-3a, suppresses platelet adhesion and activation. In nucleated cells, ß-catenin, the key downstream effector of this pathway, is a dual function protein, regulating the coordination of gene transcription and cell-cell adhesion. The specific role of ß-catenin in the anucleate platelet however remains elusive. Here, a label-free quantitative proteomic analysis of ß-catenin immunoprecipitates from human platelets is performed and nine co-immunoprecipitating proteins are identified. Three of the co-immunoprecipitating proteins (α-catenin-1, cadherin-6, and ß-catenin-interacting protein 1) are common to both resting and activated conditions. Bioinformatics analysis of proteomics data reveal a strong association of the dataset with both cadherin adherens junctions and regulators of WNT signaling. It is then verified that platelet ß-catenin and cadherin-6 interact and that this interaction is regulated by the activation state of the platelet. Taken together, this proteomics study suggests a novel role for ß-catenin in human platelets where it interacts with platelet cadherins and associated junctional proteins.

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults.

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL-1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

Dynamic Protein Interactions of the Polycomb Repressive Complex 2 during Differentiation of Pluripotent Cells.

Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, each of which are associated with gene repression. Although three subunits, EZH1/2, SUZ12, and EED, form the catalytic core of PRC2, a wider group of proteins associate with low stoichiometry. This raises the question of whether dynamic variation of the PRC2 interactome results in alternative forms of the complex during differentiation. Here we compared the physical interactions of PRC2 in undifferentiated and differentiated states of NTERA2 pluripotent embryonic carcinoma cells. Label-free quantitative proteomics was used to assess endogenous immunoprecipitation of the EZH2 and SUZ12 subunits of PRC2. A high stringency data set reflecting the endogenous state of PRC2 was produced that included all previously reported core and associated PRC2 components, and several novel interacting proteins. Comparison of the interactomes obtained in undifferentiated and differentiated cells revealed candidate proteins that were enriched in complexes isolated from one of the two states. For example, SALL4 and ZNF281 associate with PRC2 in pluripotent cells, whereas PCL1 and SMAD3 preferentially associate with PRC2 in differentiating cells. Analysis of the mRNA and protein levels of these factors revealed that their association with PRC2 correlated with their cell state-specific expression. Taken together, we propose that dynamic changes to the PRC2 interactome during differentiation may contribute to directing its activity during cell fate transitions.

Peptidomic screening of bitter and nonbitter casein hydrolysate fractions for insulinogenic peptides.

Sodium caseinate hydrolysates (NaCaH) contain biologically active peptides that can positively influence human health. However, their intense bitterness hinders their inclusion in food products. To our knowledge, no studies have investigated whether a correlation between bitterness and bioactivity exists in NaCaH, so it is not yet known what effect selective removal of bitterness has on NaCaH bioactivity. A deeper understanding of the physicochemical characteristics affecting both bitterness and bioactivity is therefore needed. The aim of this study was to use in silico analysis to elucidate the relationship between bitterness and bioactivity of the insulinogenic NaCaH. The NaCaH fractions were generated by membrane filtration and flash chromatography and were subsequently evaluated for bitterness by a sensory panel. In this present study, peptidomic and bioinformatic processing of these NaCaH fractions allowed for the identification of insulinogenic peptides as well as other literature-identified peptides in each of the fractions. The results showed that the most bitter fraction contained the highest abundance of insulinogenic peptides, whereas another bitter fraction contained the highest abundance of other literature-identified bioactive peptides exhibiting angiotensin-converting enzyme-inhibition activity. Although some bioactive peptides were identified in the least bitter fractions, the abundance of these peptides was very low. These observations show a correlation between bitter taste and bioactivity, highlighting potential complications in removing bitterness while maintaining bioactivity. However, as the most bitter fraction contained the highest abundance of insulinogenic peptides, there is potential for using a lower dose of this enriched bioactive fraction to exert health benefits. The second most bitter fraction contained a very low abundance of insulinogenic peptides and other bioactive peptides. Therefore, removal of this fraction could reduce the NaCaH product's bitterness without significantly altering overall bioactive potential.

Comparative proteomic analysis of trophoblast cell models reveals their differential phenotypes, potential uses, and limitations.

Trophoblastic cell lines are widely used in in vitro studies of placental function as a surrogate for primary trophoblasts. To date, no reference proteomics dataset exists to directly compare the shared and unique characteristics of these cells. Here, we performed comparative proteomic profiling of the BeWo and HTR8/SVneo cell lines using label-free quantitative MS. A total of 1557 proteins were identified, which included 338 uniquely attributed to BeWo cells, and a further 304 specifically identified in HTR8/SVneo cells. Raw data are available via ProteomeXchange, identifier PDX005045. Of the 915 proteins expressed by both cell lines, 105 were of higher abundance in BeWo cells, while 199 proteins had a significantly higher expression in HTR8/SVneo cells. Comparative GO of unique and upregulated proteins revealed principal differences in cell junction/adhesion, catenin complex, spindle and microtubule associated complex, as well as cell differentiation. Our data indicate that BeWo cells express an epithelial proteome more characteristic of villous trophoblasts, whereas HTR8/SVneo cells embrace a mesenchymal phenotype, more characteristic of extravillous trophoblasts. This novel comparative proteomic profiling of these trophoblastic cell lines provides a useful platform for future investigations of placental function.

The Effects of Prenatal Iron Deficiency and Risperidone Treatment on the Rat Frontal Cortex: A Proteomic Analysis.

Prenatal iron deficiency (pID) has been described to increase the risk for neurodevelopmental disorders such as autism and schizophrenia; however, the precise molecular mechanisms are still unknown. Here, we utilized high-throughput MS to examine the proteomic effects of pID in adulthood on the rat frontal cortex area (FCA). In addition, the FCA proteome was examined in adulthood following risperidone treatment in adolescence to see if these effects could be prevented. We identified 1501 proteins of which 100 were significantly differentially expressed in the FCA at postnatal day 90. Pathway analysis of proteins affected by pID revealed changes in metabolic processes, including the tricyclic acid cycle, mitochondrial dysfunction, and P13K/Akt signaling. Interestingly, most of these protein changes were not present in the adult pID offspring who received risperidone in adolescence. Considering the link between pID and several neurodevelopmental disorders such as autism and schizophrenia these presented results bring new perspectives to understand the role of iron in metabolic pathways and provide novel biomarkers for future studies of pID.

SerpinI2 (pancpin) is an inhibitory serpin targeting pancreatic elastase and chymotrypsin.

SerpinI2/Pancpin/MEPI is a 46kDa member of the serpin (serine protease inhibitor) superfamily. It is downregulated in pancreatic and breast cancer, and associated with acinar cell apoptosis and pancreatic insufficiency when absent in mice. However, the target protease and protein properties of serpinI2 are previously uncharacterised. We have expressed and purified recombinant serpin I2 in E. coli. The protein exhibited thermal instability typical of inhibitory serpins, which was lost following RCL cleavage. SerpinI2 did not inhibit trypsin, but was found to inhibit pancreatic chymotrypsin and elastase with Kass values >105M-1s-1, and with stoichiometry of inhibition of 1.4 and 1.7 respectively. Mutagenesis of the predicted critical hinge region residue Ser344 abolished inhibitory activity, and a cleavage site C-terminal to Met358 was identified. The protein is also prone to polymerisation/aggregation at 45°C, a characteristic of serpins associated with disease. This study therefore reveals a function for serpinI2 and supports the hypothesis that this protein can protect pancreatic cells from prematurely activated zymogens.