Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of
signal transduction. Here we describe, for the first time, the application of ...the tandem affinity purification (TAP) and LC-MS
method to the characterization of protein complexes from transgenic mice. The TAP strategy developed in transgenic mice allows
the emplacement of complexes in their physiological environment in contact with proteins that might only be specifically expressed
in certain tissues while simultaneously ensuring the right stoichiometry of the TAP protein versus their binding partners and represents a novelty in proteomics approaches used so far. Mouse lines expressing TAP-tagged 14-3-3ζ
protein were generated, and protein interactions were determined. 14-3-3 proteins are general regulators of cell signaling
and represent up to 1% of the total brain protein. This study allowed the identification of almost 40 novel 14-3-3ζ-binding
proteins. Biochemical and functional characterization of some of these interactions revealed new mechanisms of action of 14-3-3ζ
in several signaling pathways, such as glutamate receptor signaling via binding to homer homolog 3 (Homer 3) and in cytoskeletal
rearrangements and spine morphogenesis by binding and regulating the activity of the signaling complex formed by G protein-coupled
receptor kinase-interactor 1 (GIT1) and p21-activated kinase-interacting exchange factor β (βPIX).
Herein we describe the design and synthesis of a dual active histone deacetylase (HDAC)/bromodomain and extra terminal (BET) small molecule tool inhibitor, DUAL946 ( 1 ). Exploiting our extensive ...epigenetic toolbox, we achieved the functionalisation of a BET active tetrahydroquinoline (THQ) core, with a hydroxamic acid HDAC inhibitor (HDACi) motif. Dual inhibition of BET and HDAC proteins was confirmed by in vitro biochemical and biophysical testing and through chemoproteomic competition experiments in cell lysates. This activity was translated into potent cellular activity in both immune and cancer cells.
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.
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.
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.
We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MARKing tau for tangles and toxicity Drewes, Gerard
Trends in biochemical sciences (Amsterdam. Regular ed.),
10/2004, Letnik:
29, Številka:
10
Journal Article
Recenzirano
In healthy neurons, tau proteins regulate microtubule function in the axon. In the brains of individuals with Alzheimer's disease, tau is hyperphosphorylated and aggregated into intraneuronal ...deposits called neurofibrillary tangles (NFTs). Hyperphosporylation dislodges tau from the microtubule surface, potentially resulting in compromised axonal integrity and the accumulation of toxic tau peptides. Recent biochemical and animal model studies have re-evaluated tau phosphorylation and other aspects of neurofibrillar pathology. The results indicate that phosphorylation of tau's microtubule-binding domain by the protein kinase MARK primes tau for hyperphosphorylation by the kinases GSK-3 and Cdk5, which in turn triggers the aggregation of tau into filaments and tangles. Toxic consequences for the neuron might be exacerbated by tangle formation but are already evident during the early steps of the process.
The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited by a number of drugs. However, there has been little success in developing drugs that target ...folate metabolism in the kinetoplastids. Despite compounds being identified which show significant inhibition of the parasite enzymes, this activity does not translate well into cellular and animal models of disease. Understanding to which enzymes antifolates bind under physiological conditions and how this corresponds to the phenotypic response could provide insight on how to target the folate pathway in these organisms. To facilitate this, we have adopted a chemical proteomics approach to study binding of compounds to enzymes of folate metabolism. Clinical and literature antifolate compounds were immobilized onto resins to allow for “pull down” of the proteins in the “folateome”. Using competition studies, proteins, which bind the beads specifically and nonspecifically, were identified in parasite lysate (Trypanosoma brucei and Leishmania major) for each antifolate compound. Proteins were identified through tryptic digest, tandem mass tag (TMT) labeling of peptides followed by LC-MS/MS. This approach was further exploited by creating a combined folate resin (folate beads). The resin could pull down up to 9 proteins from the folateome. This information could be exploited in gaining a better understanding of folate metabolism in kinetoplastids and other organisms.
Comprehensive selectivity profiling is key for the development of safe drugs. Few current methods are capable of profiling large compound sets against entire target classes. The new EnPlex method ...expands the existing toolbox, allowing for the surveying of the serine hydrolase enzyme family. A study by Bachovchin et al that developed this method is discussed.
Chemical and Pathway Proteomics Kruse, Ulrich; Bantscheff, Marcus; Drewes, Gerard ...
Molecular & cellular proteomics,
October 2008, 20081001, 2008-10-00, Letnik:
7, Številka:
10
Journal Article
Recenzirano
Odprti dostop
In recent years mass spectrometry-based proteomics has moved beyond a mere quantitative description of protein expression levels and their possible correlation with disease or drug action. Impressive ...progress in LC-MS instrumentation together with the availability of new enabling tools and methods for quantitative proteome analysis and for identification of posttranslational modifications has triggered a surge of chemical and functional proteomics studies dissecting mechanisms of action of cancer drugs and molecular mechanisms that modulate signal transduction pathways. Despite the tremendous progress that has been made in the field, major challenges, relating to sensitivity, dynamic range, and throughput of the described methods, remain. In this review we summarize recent advances in LC-MS-based approaches and their application to cancer drug discovery and to studies of cancer-related pathways in cell culture models with particular emphasis on mechanistic studies of drug action in these systems. Moreover we highlight the emerging utility of pathway and chemical proteomics techniques for translational research in patient tissue.
Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic ...understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.
We extended thermal proteome profiling to detect transmembrane protein-small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we ...observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell-receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1.
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