Proteomics strategy for quantitative protein interaction profiling in cell extracts

Sharma, Kirti; Weber, Christoph K.; Bairlein, Michaela; Greff, Zoltán; Kéri, Gÿorgý; Cox, Jürgen; Olsen, Jesper V.; Daub, Henrik

doi:10.1038/nmeth.1373

Cited by 136 publications

(143 citation statements)

References 27 publications

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“…Quantification of proteins can be achieved by several nonradioactive isotope labeling approaches, notably by stable isotope labeling and by stable isotope tagging. The former method affords protein labeling by the incorporation of isotopically enriched constituent amino acids in culture (77) or by in vivo metabolic labeling in whole organisms (78). Both metabolic labeling and isobaric chemical tagging are generally capable of accurate, precise, and reproducible quantification capable of deep proteome coverage (79).…”

Section: Resultsmentioning

confidence: 99%

A Profile of an Endosymbiont-enriched Fraction of the Coral Stylophora pistillata Reveals Proteins Relevant to Microbial-Host Interactions

Weston

Dunlap

Shick

et al. 2012

Molecular & Cellular Proteomics

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This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal "bleaching" stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiontenriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure.

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Section: Resultsmentioning

confidence: 99%

A Profile of an Endosymbiont-enriched Fraction of the Coral Stylophora pistillata Reveals Proteins Relevant to Microbial-Host Interactions

Weston

Dunlap

Shick

et al. 2012

Molecular & Cellular Proteomics

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“…Determination of K d values for tubastatin were performed as described in ref. 56. The details of K d value calculation is supplied in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Histone deacetylase 10 promotes autophagy-mediated cell survival

Oehme

Linke

Böck

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

184

212

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Tumor cells activate autophagy in response to chemotherapyinduced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome.drug resistance | HDAC inhibitor | childhood tumors A utophagy is an evolutionarily highly conserved process that can be induced by metabolic or therapeutic stress, such as DNA damage-inducing drugs (1). The two dominant types of autophagy are macroautophagy and chaperone-mediated autophagy (CMA) (2). Macroautophagy is regulated by autophagyrelated genes (ATGs), including beclin-1 (ATG6) and microtubule-associated protein 1 light chain 3 (LC3) (ATG8), and involves the sequestration of cytoplasmic components within a double-membrane structure called the autophagosome and successive delivery to lysosomes for degradation (reviewed in ref.3). CMA targets specific cytosolic proteins to the lysosomes for protein degradation (4). During CMA, the cytosolic chaperone heat shock cognate (Hsc)70 binds proteins targeted for degradation and translocates them to the lysosomes (5), where they bind to the substrate protein receptor lysosome-associated membrane protein type 2A (LAMP-2A) (6).Inhibition of histone deacetylases (HDACs) by HDAC inhibitors (HDACis) have been shown to cause significant anti-tumor effects, including cell-cycle arrest, differentiation, and apoptosis, in a broad spectrum of hematologic and solid tumors (reviewed in ref. 7). The efficacy of HDACis are currently being evaluated for treating various cancers in clinical trials (7-9). Recent research carried out in several tumor cell lines has shown that apoptosis induced by HDACis i...

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“…After a 30-min preincubation with inhibitor on ice, kinase reactions were started by ATP addition and performed for 10 min at 30°C in a final volume of 25 l. Phosphate incorporation and IC 50 values were determined as described (23).…”

Section: Methodsmentioning

confidence: 99%

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

Oppermann¹,

Grundner-Culemann²,

Kumar³

et al. 2012

Molecular & Cellular Proteomics

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Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinasesubstrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates. Molecular & Cellular Proteomics 11: 10.1074/mcp.O111.012351, 1-12, 2012.Reversible protein phosphorylation by protein kinases represents a key control mechanism in signal transmission and controls nearly all aspects of cellular physiology. Quantitative proteomics approaches that incorporate techniques such as stable isotope labeling by amino acids in cell culture (SILAC), 1 phosphopeptide fractionation and enrichment by strong cation exchange (SCX), and ion metal affinity chromatography (IMAC) together with sensitive high resolution MS analysis and automated peptide identification and quantification have made it possible to monitor phosphorylation-based signaling on a global scale (1-4). Because signaling networks are defined by the underlying kinase-substrate relationships, systematic approaches are required for the comprehensive and confident assignment of cellular kinase substrates (5). To identify cellular substrates, the catalytic activity of a kinase of interest needs to be rapidly regulated to capture a high fraction of direct phosphorylation events. This implies that protein kinase ablation by genetic knockout or RNA interference can be of limited utility, because of secondary changes that can accumulate during the time required for cellular kinase depletion (3, 5). In contrast, pharmacological interference by small molecules allows for rapid modulation of kinase activity and should therefore enable unbiased monitoring of signaling perturbations when combined with advanced MS-based proteomics. S...

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Proteomics strategy for quantitative protein interaction profiling in cell extracts

Cited by 136 publications

References 27 publications

A Profile of an Endosymbiont-enriched Fraction of the Coral Stylophora pistillata Reveals Proteins Relevant to Microbial-Host Interactions

A Profile of an Endosymbiont-enriched Fraction of the Coral Stylophora pistillata Reveals Proteins Relevant to Microbial-Host Interactions

Histone deacetylase 10 promotes autophagy-mediated cell survival

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

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