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Bantscheff et al . use chemoproteomics to measure the affinity of small molecules for megadalton protein complexes in cell extracts. Differences in the selectivity of HDAC inhibitors observed when native HDAC complexes are compared with their purified catalytic subunits suggest the limitations of using isolated recombinant proteins in certain drug screens. The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.

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.

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α triggers a signalling cascade, converging on the activation of the transcription factor NF-κB, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-α/NF-κB pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-α/NF-κB pathway and is generally applicable to other pathways relevant to human disease.

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.

Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.

Abstract Cerebral palsy (CP) is a severely debilitating condition. The underlying insult results in irreversible damage to the central nervous system, treatment is essentially palliative. Botulinum toxin type-A (BTX/A) Botox® Allergan, Inc. injections have been found to be effective palliation for dynamic spasticity in CP. There is a paucity of data regarding the maximum dose tolerated in the pediatric population. The present study examines the safety and efficacy of BTX/A therapy at up to 40 U/kg. Twenty-two patients were treated from 1995 to 1999. The population included four adults and 18 pediatric patients. Patients were in three groups: diplegia (n=12), hemiplegia (n=5), and quadriplegia (n=5). They were evaluated by the Modified Ashworth Scale. The incidence and severity of complications is also reported. The significance of these data is also commented upon. This study suggests that BTX/A therapy is safe, efficacious, and cost effective at higher doses, and should be considered as part of a comprehensive therapeutic regimen in appropriate candidates. (J Pediatr Neurol 2004; 2(2): 91–96).

Abstract Tics are a common disorder in children. Some available drugs have unacceptable side effects, and thus alternative treatments are needed. Levetiracetam is an antiepileptic drug reported to be useful for the treatment of tics. Patients age 18 years and younger with tics and Tourette syndrome were enrolled in this prospective, open-label study. Seventy patients were treated with levetiracetam as monotherapy for four years. The initial dose of levetiracetam was 250 mg/day. The dose was titrated over three weeks to 1,000 to 2,000 mg/day. Clinical outcomes were assessed with the Clinical Global Impression Scale, Yale Global Tic Severity Scale, Revised Conners’ Parent Rating Scale, and Revised Conners’ Teacher Rating Scale. After four years of treatment with levetiracetam, all 70 patients showed some improvements based on the scales used. A total of 49 patients improved with regard to behavior and school performance. Levetiracetam was generally well tolerated. Despite the limitations of open-label studies, these results suggest that levetiracetam may be useful in treating tics in children and adolescents. Given its established safety profile, levetiracetam is a candidate for further study.

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.