A Bayesian semi-parametric model for thermal proteome profiling.

Funder: Wellcome TrustThe thermal stability of proteins can be altered when they interact with small molecules, other biomolecules or are subject to post-translation modifications. Thus monitoring the thermal stability of proteins under various cellular perturbations can provide insights into protein function, as well as potentially determine drug targets and off-targets. Thermal proteome profiling is a highly multiplexed mass-spectrommetry method for monitoring the melting behaviour of thousands of proteins in a single experiment. In essence, thermal proteome profiling assumes that proteins denature upon heating and hence become insoluble. Thus, by tracking the relative solubility of proteins at sequentially increasing temperatures, one can report on the thermal stability of a protein. Standard thermodynamics predicts a sigmoidal relationship between temperature and relative solubility and this is the basis of current robust statistical procedures. However, current methods do not model deviations from this behaviour and they do not quantify uncertainty in the melting profiles. To overcome these challenges, we propose the application of Bayesian functional data analysis tools which allow complex temperature-solubility behaviours. Our methods have improved sensitivity over the state-of-the art, identify new drug-protein associations and have less restrictive assumptions than current approaches. Our methods allows for comprehensive analysis of proteins that deviate from the predicted sigmoid behaviour and we uncover potentially biphasic phenomena with a series of published datasets

Whole cell proteome regulation by microRNAs captured in a pulsed SILAC mass spectrometry approach

Since gene expression is controlled on many different levels in a cell, capturing a comprehensive snapshot of all regulatory processes is a difficult task. One possibility to monitor effective changes within a cell is to directly quantify changes in protein synthesis, which reflects the accumulative impact of regulatory mechanisms on gene expression. Pulsed stable isotope labeling by amino acids in cell culture (pSILAC) has been shown to be a viable method to investigate de novo protein synthesis on a proteome-wide scale (Schwanhausser et al., Proteomics 9:205-209, 2009; Selbach et al., Nature 455:58-63, 2008). One application of pSILAC is to study the regulation of protein expression by microRNAs. Here, we describe how pSILAC in conjunction with shotgun mass spectrometry can assess differences in the protein profile between cells transfected with a microRNA and non-transfected cells

NQO2 is a reactive oxygen species generating off-target for acetaminophen

[Image: see text] The analgesic and antipyretic compound acetaminophen (paracetamol) is one of the most used drugs worldwide. Acetaminophen overdose is also the most common cause for acute liver toxicity. Here we show that acetaminophen and many structurally related compounds bind quinone reductase 2 (NQO2) in vitro and in live cells, establishing NQO2 as a novel off-target. NQO2 modulates the levels of acetaminophen derived reactive oxygen species, more specifically superoxide anions, in cultured cells. In humans, NQO2 is highly expressed in liver and kidney, the main sites of acetaminophen toxicity. We suggest that NQO2 mediated superoxide production may function as a novel mechanism augmenting acetaminophen toxicity

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2.

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines

Genetic and Proteomic Approaches to Identify Cancer Drug Targets

While target-based small-molecule discovery has taken centre-stage in the pharmaceutical industry, there are many cancer-promoting proteins not easily addressed with a traditional target-based screening approach. In order to address this problem, as well as to identify modulators of biological states in the absence of knowing the protein target of the state switch, alternative phenotypic screening approaches, such as gene expression-based and high-content imaging, have been developed. With this renewed interest in phenotypic screening, however, comes the challenge of identifying the binding protein target(s) of small-molecule hits. Emerging technologies have the potential to improve the process of target identification. In this review, we discuss the application of genomic (gene expression-based), genetic (short hairpin RNA and open reading frame screening), and proteomic approaches to protein target identification

Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics

<p>Abstract</p> <p>Background</p> <p>The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level.</p> <p>Methods</p> <p>We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ("kinobeads"). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure.</p> <p>Results</p> <p>We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain.</p> <p>Conclusions</p> <p>We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications.</p

Cell Cycle-Related Cyclin B1 Quantification

To obtain non-relative measures of cell proteins, purified preparations of the same proteins are used as standards in Western blots. We have previously quantified SV40 large T antigen expressed over a several fold range in different cell lines and correlated the average number of molecules to average fluorescence obtained by cytometry and determined cell cycle phase related expression by calculation from multi-parametric cytometry data. Using a modified approach, we report quantification of endogenous cyclin B1 and generation of the cell cycle time related expression profile.Recombinant cyclin B1 was purified from a baculovirus lysate using an antibody affinity column and concentrated. We created fixed cell preparations from nocodazole-treated (high cyclin B1) and serum starved (low cyclin B1) PC3 cells that were either lyophilized (for preservation) or solubilized. The lysates and purified cyclin B1 were subjected to Western blotting; the cell preparations were subjected to cytometry, and fluorescence was correlated to molecules. Three untreated cell lines (K562, HeLa, and RKO) were prepared for cytometry without lyophilization and also prepared for Western blotting. These were quantified by Western blotting and by cytometry using the standard cell preparations.The standard cell preparations had 1.5 x 10(5) to 2.5 x 10(6) molecules of cyclin B1 per cell on average (i.e., 16-fold range). The average coefficient of variation was 24%. Fluorescence varied 12-fold. The relationship between molecules/cell (Western blot) and immunofluorescence (cytometry) was linear (r(2) = 0.87). Average cyclin B1 levels for the three untreated cell lines determined by Western blotting and cytometry agreed within a factor of 2. The non-linear rise in cyclin B1 in S phase was quantified from correlated plots of cyclin B1 and DNA content. The peak levels achieved in G2 were similar despite differences in lineage, growth conditions, and rates of increase through the cell cycle (range: 1.6-2.2 x 10(6) molecules per cell).Net cyclin B1 expression begins in G1 in human somatic cells lines; increases non-linearly with variation in rates of accumulation, but peaks at similar peak values in different cell lines growing under different conditions. This suggests tight quantitative end point control

Tyrosine kinase inhibitor therapy-induced changes in humoral immunity in patients with chronic myeloid leukemia

Purpose Tyrosine kinase inhibitors (TKIs) have well-characterized immunomodulatory effects on T and NK cells, but the effects on the humoral immunity are less well known. In this project, we studied TKI-induced changes in B cell-mediated immunity. Methods We collected peripheral blood (PB) and bone marrow (BM) samples from chronic myeloid leukemia (CML) patients before and during first-line imatinib (n = 20), dasatinib (n = 16), nilotinib (n = 8), and bosutinib (n = 12) treatment. Plasma immunoglobulin levels were measured, and different B cell populations in PB and BM were analyzed with flow cytometry. Results Imatinib treatment decreased plasma IgA and IgG levels, while dasatinib reduced IgM levels. At diagnosis, the proportion of patients with IgA, IgG, and IgM levels below the lower limit of normal (LLN) was 0, 11, and 6% of all CML patients, respectively, whereas at 12 months timepoint the proportions were 6% (p = 0.13), 31% (p = 0.042) and 28% (p = 0.0078). Lower initial Ig levels predisposed to the development of hypogammaglobulinemia during TKI therapy. Decreased Ig levels in imatinibtreated patients were associated with higher percentages of immature BM B cells. The patients, who had low Ig levels during the TKI therapy, had significantly more frequent minor infections during the follow-up compared with the patients with normal Ig values (33% vs. 3%, p = 0.0016). No severe infections were reported, except recurrent upper respiratory tract infections in one imatinib-treated patient, who developed severe hypogammaglobulinemia. Conclusions TKI treatment decreases plasma Ig levels, which should be measured in patients with recurrent infections.Peer reviewe