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Abstract withdrawn to prevent dissemination of unpublished results.

Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound. Proteomics is often used to map protein-drug interactions but identifying a drug’s protein targets along with the binding interfaces has not been achieved yet. Here, the authors integrate limited proteolysis and machine learning for the proteome-wide mapping of drug protein targets and binding sites.

Many intra- and extracellular signals induce structural changes in proteins. Schopper et al. , describe a limited proteolysis–based mass spectrometry (LiP-MS) approach to characterizing these changes at a proteome-wide scale. Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.

Organisms have to adapt to changes in their environment. Cellular adaptation requires sensing, signalling and ultimately the activation of cellular programs. Metabolites are environmental signals that are sensed by proteins, such as metabolic enzymes, protein kinases and nuclear receptors. Recent studies have discovered novel metabolite sensors that function as gene regulatory proteins such as chromatin associated factors or RNA binding proteins. Due to their function in regulating gene expression, metabolite-induced allosteric control of these proteins facilitates a crosstalk between metabolism and gene expression. Here we discuss the direct control of gene regulatory processes by metabolites and recent progresses that expand our abilities to systematically characterize metabolite-protein interaction networks. Obtaining a profound map of such networks is of great interest for aiding metabolic disease treatment and drug target identification.

Graphical Abstract While informative, protein amounts and physical protein associations do not provide a full picture of protein function. This Commentary highlights the potential of structural and stability proteomic technologies to derive new insights in biology and medicine.

Obesity is associated with increased risk and worse prognosis of many tumours including those of the breast and of the esophagus. Adipokines released from the peritumoural adipose tissue promote the metastatic potential of cancer cells, suggesting the existence of a crosstalk between the adipose tissue and the surrounding tumour. Mitochondrial Ca 2+ signaling contributes to the progression of carcinoma of different origins. However, whether adipocyte-derived factors modulate mitochondrial Ca 2+ signaling in tumours is unknown. Here, we show that conditioned media derived from adipose tissue cultures (ADCM) enriched in precursor cells impinge on mitochondrial Ca 2+ homeostasis of target cells. Moreover, in modulating mitochondrial Ca 2+ responses, a univocal crosstalk exists between visceral adipose tissue-derived preadipocytes and esophageal cancer cells, and between subcutaneous adipose tissue-derived preadipocytes and triple-negative breast cancer cells. An unbiased metabolomic analysis of ADCM identified creatine and creatinine for their ability to modulate mitochondrial Ca 2+ uptake, migration and proliferation of esophageal and breast tumour cells, respectively.

MitoK ATP is a channel of the inner mitochondrial membrane that controls mitochondrial K + influx according to ATP availability. Recently, the genes encoding the pore-forming (MITOK) and the regulatory ATP-sensitive (MITOSUR) subunits of mitoK ATP were identified, allowing the genetic manipulation of the channel. Here, we analyzed the role of mitoK ATP in determining skeletal muscle structure and activity. Mitok −/− muscles were characterized by mitochondrial cristae remodeling and defective oxidative metabolism, with consequent impairment of exercise performance and altered response to damaging muscle contractions. On the other hand, constitutive mitochondrial K + influx by MITOK overexpression in the skeletal muscle triggered overt mitochondrial dysfunction and energy default, increased protein polyubiquitination, aberrant autophagy flux, and induction of a stress response program. MITOK overexpressing muscles were therefore severely atrophic. Thus, the proper modulation of mitoK ATP activity is required for the maintenance of skeletal muscle homeostasis and function.

Transcriptional memory, by which cells respond faster to repeated stimuli, is key for cellular adaptation and organism survival. Chromatin organization has been shown to play a role in the faster response of primed cells. However, the contribution of post-transcriptional regulation is not yet explored. Here we perform a genome-wide screen to identify novel factors modulating transcriptional memory in S. cerevisiae in response to galactose. We find that depletion of the nuclear RNA exosome increases GAL1 expression in primed cells. Our work shows that gene-specific differences in intrinsic nuclear surveillance factor association can enhance both gene induction and repression in primed cells. Finally, we show that primed cells present altered levels of RNA degradation machinery and that both nuclear and cytoplasmic mRNA decay modulate transcriptional memory. Our results demonstrate that mRNA post-transcriptional regulation, and not only transcription regulation, should be considered when investigating gene expression memory. Transcriptional memory is key for cellular adaptation. Here the authors show that differences in mRNA stability and mRNA degradation machinery between naïve and primed cells facilitate faster gene expression response to repeated stimuli.

Adult-onset Still’s disease (AOSD) is a systemic inflammatory condition of unknown aetiology characterized by typical episodes of spiking fever, evanescent rash, arthralgia, leukocytosis and hyperferritinemia. Given the lack of data in Italian series, we promote a multicentric data collection to characterize the clinical phenotype of Italian patients with AOSD. Data from 245 subjects diagnosed with AOSD were collected by 15 centres between March and May 2013. The diagnosis was made following Yamaguchi’s criteria. Data regarding clinical manifestations, laboratory features, disease course and treatments were reported and compared with those presented in other published series of different ethnicity. The most frequent features were the following: arthritis (93 %), pyrexia (92.6 %), leukocytosis (89 %), negative ANA (90.4 %) and neutrophilia (82 %). As compared to other North American, North European, Middle Eastern and Far Eastern cohorts, Italian data show differences in clinical and laboratory findings. Regarding the treatments, in 21.9 % of cases, corticosteroids and traditional DMARDs have not been able to control the disease while biologics have been shown to be effective in 48 to 58 patients. This retrospective work summarizes the largest Italian multicentre series of AOSD patients and presents clinical and laboratory features that appear to be influenced by the ethnicity of the affected subjects.