Proteomic Mapping of the Interactome of KRAS Mutants Identifies New Features of RAS Signalling Networks and the Mechanism of Action of Sotorasib

RAS proteins are key regulators of cell signalling and control different cell functions including cell proliferation, differentiation, and cell death. Point mutations in the genes of this family are common, particularly in KRAS. These mutations were thought to cause the constitutive activation of KRAS, but recent findings showed that some mutants can cycle between active and inactive states. This observation, together with the development of covalent KRASG12C inhibitors, has led to the arrival of KRAS inhibitors in the clinic. However, most patients develop resistance to these targeted therapies, and we lack effective treatments for other KRAS mutants. To accelerate the development of RAS targeting therapies, we need to fully characterise the molecular mechanisms governing KRAS signalling networks and determine what differentiates the signalling downstream of the KRAS mutants. Here we have used affinity purification mass-spectrometry proteomics to characterise the interactome of KRAS wild-type and three KRAS mutants. Bioinformatic analysis associated with experimental validation allows us to map the signalling network mediated by the different KRAS proteins. Using this approach, we characterised how the interactome of KRAS wild-type and mutants is regulated by the clinically approved KRASG12C inhibitor Sotorasib. In addition, we identified novel crosstalks between KRAS and its effector pathways including the AKT and JAK-STAT signalling modules.European Commission Horizon 2020Science Foundation Irelan

Bioactive hydrolysates from bovine blood globulins: Generation, characterisation, and in silico prediction of toxicity and allergenicity

peer-reviewedTwo protein fractions rich in γ-globulins (FI) and α- and β-globulins (FII) were generated from bovine blood and hydrolysed with the enzyme papain. The generated hydrolysates showed in vitro angiotensin-I-converting enzyme (ACE-I), renin, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. A total of 626 and 2246 peptides were identified by LC-MS/MS from the 1 kDa fractions of FI and FII, and the potential toxicity and allergenicity of these peptides were assessed in silico using three independent predictive approaches. All of the peptides identified from the bioactive blood protein fractions FI and FII were predicted to be non-toxic. However, 72 peptides from FI and 492 peptides from FII were identified as potential allergens with at least two predictive approaches. Results suggested that papain hydrolysates of FI and FII contain potential allergenic peptides, and those products containing such hydrolysates should be labelled correctly in line with Food and Consumer legislation – Regulation (EU) No.1169/2011

Proteomics Strategy for Identifying Candidate Bioactive Proteins in Complex Mixtures: Application to the Platelet Releasate

Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples

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

In silico and in vitro analyses of the angiotensin-I converting enzyme inhibitory activity of hydrolysates generated from crude barley (Hordeum vulgare) protein concentrates

peer-reviewedAngiotensin-I-converting enzyme (ACE-I) plays a key role in control of hypertension, and type-2 diabetes mellitus, which frequently co-exist. Our current work utilised in silico methodologies and peptide databases as tools for predicting release of ACE-I inhibitory peptides from barley proteins. Papain was the enzyme of choice, based on in silico analysis, for experimental hydrolysis of barley protein concentrate, which was performed at the enzyme’s optimum conditions (60 °C, pH 6.0) for 24 h. The generated hydrolysate was subjected to molecular weight cut-off (MWCO) filtration, following which the non-ultrafiltered hydrolysate (NUFH), and the generated 3 kDa and 10 kDa MWCO filtrates were assessed for their in vitro ACE-I inhibitory activities. The 3 kDa filtrate (1 mg/ml), that demonstrated highest ACE-I inhibitory activity of 70.37%, was characterised in terms of its peptidic composition using mass spectrometry and 1882 peptides derived from 61 barley proteins were identified, amongst which 15 peptides were selected for chemical synthesis based on their predicted ACE-I inhibitory properties. Of the synthesized peptides, FQLPKF and GFPTLKIF were most potent, demonstrating ACE-I IC50 values of 28.2 μM and 41.2 μM respectively.Food Institutional Research Measur

ApoE elevation is associated with the persistence of psychotic experiences from age 12 to age 18: Evidence from the ALSPAC birth cohort

Apolipoproteins, which play important roles in lipid metabolism, innate immunity and synaptic signalling, have been implicated in first episode psychosis and schizophrenia. This is the first study to investigate plasma apolipoprotein expression in children with psychotic experiences that persist into adulthood. Here, using semi-targeted proteomic analysis we compared plasma apolipoprotein expression levels in age 12 subjects who reported psychotic experiences at both age 12 and age 18 (n = 37) with age-matched subjects who only experienced psychotic experiences (PEs) at age 12 (n = 38). 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. We identified apoE, a protein with significant regulatory activity on cholesterol metabolism in the brain, to be significantly up regulated (p < 0.003) in those with persistent psychotic experiences. We confirmed this finding in these samples using ELISA. Our findings indicate elevated plasma apoE in age 12 children who experience PEs is associated with persistence psychotic experiences

Blood-Based Protein Changes in Childhood Are Associated With increased risk for later psychotic disorder: evidence from a nested case–control study of the ALSPAC Longitudinal Birth Cohort

The identification of early biological changes associated with the psychotic disorder (PD) is important as it may provide clues to the underlying pathophysiological mechanisms. We undertook the first proteomic profiling of blood plasma samples of children who later develop a PD. Participants were recruited from the UK Avon Longitudinal Study of Parents and Children (ALSPAC) cohort who also participated in psychiatric assessment interviews at age 18. Protein expression levels at age 11 were compared between individuals who developed PD at age 18 (n = 37) with population-based age-matched controls (n = 38). Sixty out of 181 plasma proteins profiled were found to be differentially expressed (P < .05) in children with an outcome of the PD. Thirty-four of these proteins were found to be differentially expressed following correction for multiple comparisons. Pathway analysis implicated the complement and coagulation cascade. A second, targeted proteomic approach was used to verify these findings in age 11 plasma from subjects who reported psychotic experiences at age 18 (n = 40) in comparison to age-matched controls (n = 66). Our findings indicate that the complement and coagulation system is dysregulated in the blood during childhood before the development of the PD