Systems Pharmacology Dissection of Cholesterol Regulation Reveals Determinants of Large Pharmacodynamic Variability between Cell Lines

Blattmann, Peter; Henriques, David; Zimmermann, Michael; Frommelt, Fabian; Sauer, Uwe; Sáez-Rodríguez, Julio; Aebersold, Ruedi

doi:10.1016/j.cels.2017.11.002

Cited by 25 publications

(20 citation statements)

References 71 publications

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“…To assess the localization of the phosphorylation site for each annotated spectra, the same strategy as reported in ref. 62 was used. In brief, a global false localization rate (FLR) was calculated using LuciPHOr2 30 , allowing the classification of localized and non-localized phosphopeptides.…”

Section: Methodsmentioning

confidence: 99%

Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes

et al. 2020

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Rapidly increasing availability of genomic data and ensuing identification of disease associated mutations allows for an unbiased insight into genetic drivers of disease development. However, determination of molecular mechanisms by which individual genomic changes affect biochemical processes remains a major challenge. Here, we develop a multilayered proteomic workflow to explore how genetic lesions modulate the proteome and are translated into molecular phenotypes. Using this workflow we determine how expression of a panel of disease-associated mutations in the Dyrk2 protein kinase alter the composition, topology and activity of this kinase complex as well as the phosphoproteomic state of the cell. The data show that altered protein-protein interactions caused by the mutations are associated with topological changes and affected phosphorylation of known cancer driver proteins, thus linking Dyrk2 mutations with cancer-related biochemical processes. Overall, we discover multiple mutation-specific functionally relevant changes, thus highlighting the extensive plasticity of molecular responses to genetic lesions.

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Section: Methodsmentioning

confidence: 99%

Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes

et al. 2020

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“…Furthermore, different HeLa Kyoto clones between labs were 83 reported to vary in terms of mRNA expression 18 . However, little is known about the manner by 84 which such genomic variability affects the proteomes or phenotypes of the different HeLa strains 85 between individual labs, the effect of successive passages within a stock-derived line on its 86 molecular make up, and how these would affect a biological research outcome 31,32 . 87 88 Proteins catalyze and control most biological processes.…”

Section: Introductionmentioning

confidence: 99%

Genomic, Proteomic and Phenotypic Heterogeneity in HeLa Cells across Laboratories: Implications for Reproducibility of Research Results

Liu

Mueller

et al. 2018

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1The independent reproduction of research results is a cornerstone of experimental research, yet it is 2 beset by numerous challenges, including the quality and veracity of reagents and materials. Much of 3 life science research depends on life materials, including human tissue culture cells. In this study we 4 aimed at determining the degree of variability in the molecular makeup and the ensuing phenotypic 5 consequences in commonly used human tissue culture cells. We collected 14 stock HeLa aliquots 6 from 13 different laboratories across the globe, cultured them in uniform conditions and profiled the 7 genome-wide copy numbers, mRNAs, proteins and protein turnover rates via genomic techniques 8 and SWATH mass spectrometry, respectively. We also phenotyped each cell line with respect to the 9 ability of transfected Let7 mimics to modulate Salmonella infection. 10 11We discovered significant heterogeneity between HeLa variants, especially between lines of the 12 CCL2 and Kyoto variety. We also observed progressive divergence within a specific cell line over 13 50 successive passages. From the aggregate multi-omic datasets we quantified the response of the 14 cells to genomic variability across the transcriptome and proteome. We discovered organelle-specific 15 proteome remodeling and buffering of protein abundance by protein complex stoichiometry, 16 mediated by the adaptation of protein turnover rates. By associating quantitative proteotype and 17 phenotype measurements we identified protein patterns that explained the varying response of the 18 different cell lines to Salmonella infection. 19 20Altogether the results indicate a striking degree of genomic variability, the rapid evolution of 21 genomic variability in culture and its complex translation into distinctive expressed molecular and 22 phenotypic patterns. The results have broad implications for the interpretation and reproducibility of 23 research results obtained from HeLa cells and provide important basis for a general discussion of the 24 value and requirements for communicating research results obtained from human tissue culture cells. 25 7 molecular analyses, we centrally cultured all 14 cell lines for an additional three passages in our 126 group. To minimize experimental variability, the same researcher cultured the cells using the same 127 culture media and protocol. 128 129As a starting point for the evaluation of HeLa cell heterogeneity, we measured gene copy number 130 variation (CNV) by array-CGH (aCGH). Previous studies reported genome instability within and 131 between different HeLa strains 18, 25-28 . To investigate and display the ploidy variation of the strains 132 studied we projected the CNV data onto the reference human genomic map (Figure 1b). We 133 discovered pervasive CNV differences organized by domains, large chromosomal segments, and 134 even whole chromosomes. Particularly notable were ploidy changes at Chromosomes (Chr) 1, 2, 6, 135 9, 10, 17, 19, 21, 22, and X. (Figure 1b). On average, the HeLa genome of all cells tested...

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“…Various studies had previously linked viperin to the downregulation of cholesterol biosynthesis (39, 46, 55), but the effect of viperin expression of cellular cholesterol biosynthesis had not been investigated. To examine this question, HEK293T cells were grown in reduced serum media containing lipoprotein-depleted serum to minimize uptake of exogenous cholesterol (56). Cells were transfected with either viperin or an empty vector control and grown for a further 36 h before harvesting.…”

Section: Resultsmentioning

confidence: 99%

Viperin inhibits cholesterol biosynthesis and interacts with enzymes in the cholesterol biosynthetic pathway

Grunkemeyer

Ghosh

Patel

et al. 2021

Preprint

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Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (virus inhibitory protein endoplasmic reticulum-associated, interferon-induced) is an ER membrane-associated enzyme that when expressed in response to viral infections exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. Here we have investigated the effect of viperin expression on cholesterol biosynthesis. We found that viperin expression reduces cholesterol levels by 20 to 30 % in HEK293T cells. A proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits. The two most highly enriched proteins were lanosterol synthase and squalene monooxygenase, enzymes that catalyze key steps establishing the sterol carbon skeleton. Co-immunoprecipitation experiments established that viperin, lanosterol synthase and squalene monooxygenase form a complex at the ER membrane. Co-expression of viperin was found to significantly inhibit the specific activity of lanosterol synthase in HEK293T cell lysates. Co-expression of viperin had no effect on the specific activity of squalene monooxygenase, but reduced its expression levels in the cells by approximately 30 %. Despite these inhibitory effects, co-expression of either LS or SM failed to reverse the viperin-induced depletion of cellular cholesterol levels in HEK293T cells. Our results establish a clear link between the down-regulation of cholesterol biosynthesis and viperin, although at this point the effect cannot be unambiguously attributed interactions between viperin and a specific biosynthetic enzyme.

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Systems Pharmacology Dissection of Cholesterol Regulation Reveals Determinants of Large Pharmacodynamic Variability between Cell Lines

Cited by 25 publications

References 71 publications

Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes

Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes

Genomic, Proteomic and Phenotypic Heterogeneity in HeLa Cells across Laboratories: Implications for Reproducibility of Research Results

Viperin inhibits cholesterol biosynthesis and interacts with enzymes in the cholesterol biosynthetic pathway

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