Network integration of thermal proteome profiling with multi-omics data decodes PARP inhibition.

Complex disease phenotypes often span multiple molecular processes. Functional characterization of these processes can shed light on disease mechanisms and drug effects. Thermal Proteome Profiling (TPP) is a mass-spectrometry (MS) based technique assessing changes in thermal protein stability that can serve as proxies of functional protein changes. These unique insights of TPP can complement those obtained by other omics technologies. Here, we show how TPP can be integrated with phosphoproteomics and transcriptomics in a network-based approach using COSMOS, a multi-omics integration framework, to provide an integrated view of transcription factors, kinases and proteins with altered thermal stability. This allowed us to recover consequences of Poly (ADP-ribose) polymerase (PARP) inhibition in ovarian cancer cells on cell cycle and DNA damage response as well as interferon and hippo signaling. We found that TPP offers a complementary perspective to other omics data modalities, and that its integration allowed us to obtain a more complete molecular overview of PARP inhibition. We anticipate that this strategy can be used to integrate functional proteomics with other omics to study molecular processes.

Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia.

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Short-Term Changes in Quality of Life in Patients with Advanced Lung Cancer during In-Hospital Exercise Training and Chemotherapy Treatment: A Randomized Controlled Trial.

The aim of this study was to assess the impact of exercise training on the quality of life (QoL) of patients diagnosed with stage IIIB and stage IV non-small cell lung cancer (NSCLC) compared to a passive control group (CG). The exercise-trained group (ETG) consisted of 18 patients, and the CG consisted of 8 patients. The training program in the ETG consisted of two 2-week running cycles interspersed with consecutive rounds of chemotherapy with cytostatic drugs. A comparison of the changes in the Short Form (36) Health Survey (SF-36), St. George's Respiratory Questionnaire (SGRQ), and the Functional Assessment of Cancer Therapy-Lung (FACT-L) was the primary outcome. Analysis of the results of the SGRQ and the SF-36 questionnaire did not reveal any statistically significant differences in the assessment of QoL between the examined groups. The analysis of FACT-L questionnaires showed statistically significant changes, indicating deterioration of QoL in domains describing physical well-being in the CG. Therefore, the analysis of the results of the QoL assessment did not show any significant improvements in the group of patients undergoing comprehensive exercise training, although deterioration of QoL was noted in the CG.

Halogen Atoms in the Protein-Ligand System. Structural and Thermodynamic Studies of the Binding of Bromobenzotriazoles by the Catalytic Subunit of Human Protein Kinase CK2.

Binding of a family of brominated benzotriazoles to the catalytic subunit of human protein kinase CK2 (hCK2α) was used as a model system to assess the contribution of halogen bonding to protein-ligand interaction. CK2 is a constitutively active pleiotropic serine/threonine protein kinase that belongs to the CMGC group of eukaryotic protein kinases (EPKs). Due to the addiction of some cancer cells, CK2 is an attractive and well-characterized drug target. Halogenated benzotriazoles act as ATP-competitive inhibitors with unexpectedly good selectivity for CK2 over other EPKs. We have characterized the interaction of bromobenzotriazoles with hCK2α by X-ray crystallography, low-volume differential scanning fluorimetry, and isothermal titration calorimetry. Properties of free ligands in solution were additionally characterized by volumetric and RT-HPLC measurements. Thermodynamic data indicate that the affinity increases with bromo substitution, with greater contributions from 5- and 6-substituents than 4- and 7-substituents. Except for 4,7-disubstituted compounds, the bromobenzotriazoles adopt a canonical pose with the triazole close to lysine 68, which precludes halogen bonding. More highly substituted benzotriazoles adopt many additional noncanonical poses, presumably driven by a large hydrophobic contribution to binding. Some noncanonical ligand orientations allow the formation of halogen bonds with the hinge region. Consistent with a predominantly hydrophobic interaction, the isobaric heat capacity decreases upon ligand binding, the more so the higher the substitution.

Identifying drug targets in tissues and whole blood with thermal-shift profiling.

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.

Exercise Training in Patients With Non-Small Cell Lung Cancer During In-Hospital Chemotherapy Treatment: A RANDOMIZED CONTROLLED TRIAL.

PURPOSE: The aim of this study was to perform a randomized trial to assess the impact of exercise training in patients with non-small cell lung cancer during chemotherapy on several outcomes in comparison to a control group (CG). METHODS: The exercise training group (ETG) consisted of 20 patients and the CG consisted of 10 patients. In the ETG, a 4-wk in-hospital exercise training program was performed in 2-wk cycles interspersed with consecutive rounds of chemotherapy with cytostatic drugs. The exercise training program was individualized and included warm-up, respiratory muscle exercise, training on a cycle ergometer or treadmill, and Nordic walking. CG participants were assessed before and after 6 wk of chemotherapy alone. RESULTS: Comparing pre- and post-intervention values, the ETG demonstrated an increase in 6-min walk distance (486 ± 92 vs 531 ± 103 m, P = .01). In a battery of physical performance tests: Up and Go Test (6.3 ± 1.0 vs 6.0 ± 1.1 sec, P = .01); chair stand (13.3 ± 2.8 vs 14.3 ± 3.4 repetitions, P = .001); and arm curl (18.4 ± 3.1 vs 20.4 ± 3.5 repetitions, P = .001) all improved significantly. Spirometry values also improved: FEV1 % predicted (76 ± 16 vs 84 ± 15, P = .01), FVC % predicted (87 ± 14 vs 95 ± 13, P = .01), and FEV1/FVC (73 ± 13% vs 76 ± 12%, P = .04). The exercise training was well tolerated, without any adverse events due to exercise. There were no significant improvements in the CG. CONCLUSIONS: This study suggests that planned, individualized, and supervised exercise programs in patients with advanced lung cancer during chemotherapy are a practical and beneficial intervention for enhancing mobility and physical fitness.

Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis.

Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.

Pulmonary Rehabilitation with a Stabilometric Platform after Thoracic Surgery: A Preliminary Report.

The aim of this study was to evaluate the influence of exercises on a stabilometric platform on the physical fitness and mobility of patients with lung cancer after thoracic surgery. The Experimental Group included 22, and the Control Group consisted of 21 patients. All included patients were enrolled after thoracic surgery due to lung cancer. The Experimental and Control Groups were enrolled in a 3-week in-hospital pulmonary rehabilitation program. The Experimental Group additionally performed daily 20-min training sessions on a stabilometric platform. Agility and flexibility were assessed with the Fullerton test before and after rehabilitation in both groups, and quality of life was assessed with the SF-36 questionnaire. Exercise performance stated as a distance in a 6 min walking test (6MWT) significantly improved in both groups with a medium effect size. The results of the Fullerton test indicated improvements in flexibility in both groups after the completion of the program without a significant difference between the groups and with a small effect size. In the Experimental Group, the best results were observed in the Arm curl (p = 0.0001), Chair stand (p = 0.04), Up and go (p = 0.001) and Chair sit and reach (p = 0.0001) tasks. No deterioration in the quality of life was observed in the Experimental or the Control Group after the completion of the program. Between-group analyses revealed significant differences in the Role-Physical (RP) (p = 0.020) and Mental-Health (MH) (p = 0.025) domains of the SF-36. The rehabilitation program with a stabilometric platform improved agility and flexibility of patients after thoracic surgery without an effect size or significant differences between the Experimental and Control Groups.

Click chemistry enables preclinical evaluation of targeted epigenetic therapies.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.

Condensin complexes have central roles in the three-dimensional organization of chromosomes during cell divisions, but how they interact with chromatin to promote chromosome segregation is largely unknown. Previous work has suggested that condensin, in addition to encircling chromatin fibers topologically within the ring-shaped structure formed by its SMC and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA formed by the two HEAT-repeat subunits of the Saccharomyces cerevisiae condensin complex. From detailed mapping data of the interfaces between the HEAT-repeat and kleisin subunits, we generated condensin complexes that lack one of the HEAT-repeat subunits and consequently fail to associate with chromosomes in yeast and human cells. The finding that DNA binding by condensin's HEAT-repeat subunits stimulates the SMC ATPase activity suggests a multistep mechanism for the loading of condensin onto chromosomes.

A rapidly reversible chemical dimerizer system to study lipid signaling in living cells.

Chemical dimerizers are powerful tools for non-invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small-molecule-based dimerization system and demonstrate a sufficiently fast switch-off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3-kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.

Condensin: crafting the chromosome landscape.

The successful transmission of complete genomes from mother to daughter cells during cell divisions requires the structural re-organization of chromosomes into individualized and compact structures that can be segregated by mitotic spindle microtubules. Multi-subunit protein complexes named condensins play a central part in this chromosome condensation process, but the mechanisms behind their actions are still poorly understood. An increasing body of evidence suggests that, in addition to their role in shaping mitotic chromosomes, condensin complexes have also important functions in directing the three-dimensional arrangement of chromatin fibers within the interphase nucleus. To fulfill their different functions in genome organization, the activity of condensin complexes and their localization on chromosomes need to be strictly controlled. In this review article, we outline the regulation of condensin function by phosphorylation and other posttranslational modifications at different stages of the cell cycle. We furthermore discuss how these regulatory mechanisms are used to control condensin binding to specific chromosome domains and present a comprehensive overview of condensin's interaction partners in these processes.

Effect of a static magnetic field of 7 mT on formaldehyde biodegradation in industrial wastewater from urea-formaldehyde resin production by activated sludge.

The goal of this study was to assess the efficiency of treating industrial urea-formaldehyde wastewater by activated sludge in a static magnetic field (MF) of 7 mT and the efficiency of treating the wastewater in a bioreactor not exposed to an MF. Exposure to the MF increased formaldehyde (FA) removal from industrial wastewater with an FA concentration of 1600 mg/l by 20%. The MF had also a positive effect on the efficiency of chemical oxygen demand (COD) removal, and bacteria and activated sludge biomass growth, especially when the COD loading increased rapidly. Industrial wastewater may contain up to 13000 mg FA/l. Therefore, its treatment can require the application of more than one method to ensure that the final FA concentration will be within the permissible limit. The application of an MF to enhance the biological processes may be favourable solution to this problem.

Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies.

Here we present a method to purify large amounts of highly pure and stably arrested ribosome-nascent chain complexes (RNCs) from Escherichia coli cells. It relies on the combined use of translation-arrest sequences to generate nascent polypeptides of specified length and subsequent tag purification of the RNCs. Moreover, we adapted this method for the in vivo production of RNCs with specific isotope labeling of the nascent chains for nuclear magnetic resonance (NMR) studies. This method opens therefore possibilities for a wide range of biochemical and structural studies exploring conformations of nascent chains during the early steps of protein folding and targeting.

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome.

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.

Dynamics of trigger factor interaction with translating ribosomes.

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.

The C-terminal domain of Escherichia coli trigger factor represents the central module of its chaperone activity.

In bacteria, ribosome-bound Trigger Factor assists the folding of newly synthesized proteins. The N-terminal domain (N) of Trigger Factor mediates ribosome binding, whereas the middle domain (P) harbors peptidyl-prolyl isomerase activity. The function of the C-terminal domain (C) has remained enigmatic due to structural instability in isolation. Here, we have characterized a stabilized version of the C domain (C(S)), designed on the basis of the recently solved atomic structure of Trigger Factor. Strikingly, only the isolated C(S) domain or domain combinations thereof (NC(S), PC(S)) revealed substantial chaperone activity in vitro and in vivo. Furthermore, to disrupt the C domain without affecting the overall Trigger Factor structure, we generated a mutant (Delta53) by deletion of the C-terminal 53 amino acid residues. This truncation caused the complete loss of the chaperone activity of Trigger Factor in vitro and severely impaired its function in vivo. Therefore, we conclude that the chaperone activity of Trigger Factor critically depends on its C-terminal domain as the central structural chaperone module. Intriguingly, a structurally similar module is found in the periplasmic chaperone SurA and in MPN555, a protein of unknown function. We speculate that this conserved module can exist solely or in combination with additional domains to fulfill diverse chaperone functions in the cell.

Trigger factor forms a protective shield for nascent polypeptides at the ribosome.

In prokaryotes, the ribosome-associated Trigger Factor is the first chaperone newly synthesized polypeptides encounter when they emerge from the ribosomal exit tunnel. The effects that Trigger Factor exerts on nascent polypeptides, however, remain unclear. Here we analyzed the potential of the Trigger Factor to shield nascent polypeptides at the ribosome. A set of arrested nascent polypeptides differing in origin, size, and folding status were synthesized in an Escherichia coli-based in vitro transcription/translation system and tested for sensitivity to degradation by the unspecific protease proteinase K. In the absence of Trigger Factor, nascent polypeptides exposed outside the ribosomal exit tunnel were rapidly degraded unless they were folded into a compact domain. The presence of Trigger Factor, as well as a Trigger Factor fragment lacking its peptidyl-prolyl isomerase domain, counteracted degradation of all unfolded nascent polypeptides tested. This protective function was specific for ribosome-tethered Trigger Factor, since neither non-ribosomal Trigger Factor nor the DnaK system, which cooperates with Trigger Factor in the folding process in vivo, revealed a comparable efficiency in protection. Furthermore, shielding by Trigger Factor was not restricted to short stretches of nascent chains but was evident for large, non-native nascent polypeptides exposing up to 41 kDa outside the ribosome. We suggest that Trigger Factor supports productive de novo folding by shielding nascent polypeptides on the ribosome thereby preventing untimely degradation or aggregation processes. This protected environment provided by Trigger Factor might be particularly important for large multidomain proteins to fold productively into their native states.