CDS-DB, an omnibus for patient-derived gene expression signatures induced by cancer treatment.

Patient-derived gene expression signatures induced by cancer treatment, obtained from paired pre- and post-treatment clinical transcriptomes, can help reveal drug mechanisms of action (MOAs) in cancer patients and understand the molecular response mechanism of tumor sensitivity or resistance. Their integration and reuse may bring new insights. Paired pre- and post-treatment clinical transcriptomic data are rapidly accumulating. However, a lack of systematic collection makes data access, integration, and reuse challenging. We therefore present the Cancer Drug-induced gene expression Signature DataBase (CDS-DB). CDS-DB has collected 78 patient-derived, paired pre- and post-treatment transcriptomic source datasets with uniformly reprocessed expression profiles and manually curated metadata such as drug administration dosage, sampling time and location, and intrinsic drug response status. From these source datasets, 2012 patient-level gene perturbation signatures were obtained, covering 85 therapeutic regimens, 39 cancer subtypes and 3628 patient samples. Besides data browsing, download and search, CDS-DB also supports single signature analysis (including differential gene expression, functional enrichment, tumor microenvironment and correlation analyses), signature comparative analysis and signature connectivity analysis. This provides insights into drug MOA and its heterogeneity in patients, drug resistance mechanisms, drug repositioning and drug (combination) discovery, etc. CDS-DB is available at http://cdsdb.ncpsb.org.cn/.

DCABM-TCM: A Database of Constituents Absorbed into the Blood and Metabolites of Traditional Chinese Medicine.

Traditional Chinese medicine (TCM) not only maintains the health of Asian people but also provides a great resource of active natural products for modern drug development. Herein, we developed a Database of Constituents Absorbed into the Blood and Metabolites of TCM (DCABM-TCM), the first database systematically collecting blood constituents of TCM prescriptions and herbs, including prototypes and metabolites experimentally detected in the blood, together with the corresponding detailed detection conditions through manual literature mining. The DCABM-TCM has collected 1816 blood constituents with chemical structures of 192 prescriptions and 194 herbs and integrated their related annotations, including physicochemical, absorption, distribution, metabolism, excretion, and toxicity properties, and associated targets, pathways, and diseases. Furthermore, the DCABM-TCM supported two blood constituent-based analysis functions, the network pharmacology analysis for TCM molecular mechanism elucidation, and the target/pathway/disease-based screening of candidate blood constituents, herbs, or prescriptions for TCM-based drug discovery. The DCABM-TCM is freely accessible at http://bionet.ncpsb.org.cn/dcabm-tcm/. The DCABM-TCM will contribute to the elucidation of effective constituents and molecular mechanism of TCMs and the discovery of TCM-derived drug-like compounds that are both bioactive and bioavailable.

Exploration of Target Spaces in the Human Genome for Protein and Peptide Drugs.

After decades of development, protein and peptide drugs have now grown into a major drug class in the marketplace. Target identification and validation are crucial for the discovery of protein and peptide drugs, and bioinformatics prediction of targets based on the characteristics of known target proteins will help improve the efficiency and success rate of target selection. However, owing to the developmental history in the pharmaceutical industry, previous systematic exploration of the target spaces has mainly focused on traditional small-molecule drugs, while studies related to protein and peptide drugs are lacking. Here, we systematically explore the target spaces in the human genome specifically for protein and peptide drugs. Compared with other proteins, both successful protein and peptide drug targets have many special characteristics, and are also significantly different from those of small-molecule drugs in many aspects. Based on these features, we develop separate effective genome-wide target prediction models for protein and peptide drugs. Finally, a user-friendly web server, Predictor Of Protein and PeptIde drugs' therapeutic Targets (POPPIT) (http://poppit.ncpsb.org.cn/), is established, which provides not only target prediction specifically for protein and peptide drugs but also abundant annotations for predicted targets.

CTR-DB, an omnibus for patient-derived gene expression signatures correlated with cancer drug response.

To date, only some cancer patients can benefit from chemotherapy and targeted therapy. Drug resistance continues to be a major and challenging problem facing current cancer research. Rapidly accumulated patient-derived clinical transcriptomic data with cancer drug response bring opportunities for exploring molecular determinants of drug response, but meanwhile pose challenges for data management, integration, and reuse. Here we present the Cancer Treatment Response gene signature DataBase (CTR-DB, http://ctrdb.ncpsb.org.cn/), a unique database for basic and clinical researchers to access, integrate, and reuse clinical transcriptomes with cancer drug response. CTR-DB has collected and uniformly reprocessed 83 patient-derived pre-treatment transcriptomic source datasets with manually curated cancer drug response information, involving 28 histological cancer types, 123 drugs, and 5139 patient samples. These data are browsable, searchable, and downloadable. Moreover, CTR-DB supports single-dataset exploration (including differential gene expression, receiver operating characteristic curve, functional enrichment, sensitizing drug search, and tumor microenvironment analyses), and multiple-dataset combination and comparison, as well as biomarker validation function, which provide insights into the drug resistance mechanism, predictive biomarker discovery and validation, drug combination, and resistance mechanism heterogeneity.

Characterization and immunoregulatory activity of two polysaccharides from the root of Ilex asprella.

The polysaccharides obtained from the root of Ilex asprella, namely IAPS-1 and IAPS-2, with immunoregulatory activity were studied. Two polysaccharides were isolated and purified by Cellulose DEAE-52 and Sephadex columns. The structure of IAPS-1 was elucidated as 1,6-linked α-d-glucopyranosyl main chain with branch chain substituted at C-2 and/or C-4 position. For IAPS-2, the backbone is composed of 1, 4-linked α-d-glucose, galactose and rhamnose, and branched chains consists of arabinose, rhamnose and galacturonic acid, as confirmed by partial acid hydrolysis and NMR study. Further, the immunoregulatory activity of IAPS-1 and IAPS-2 was tested with the murine macrophages. Particularly, IAPS-2 polysaccharide can more effectively enhance the secretion of major inflammatory cytokines in macrophages, such as TNF-α, IL-1ß, IL-12, compared with IAPS-1.

Water solubility is essential for fluorescent probes to image hypochlorous acid in live cells.

We synthesised four probes and compared their HClO-detecting ability in different solvents. The data showed that only hydrophilic probes could sensitively and accurately detect HClO in live cells. Meanwhile, the addition of organic solvents, as is commonly practised, weakens the oxidising capacity of HClO and thus generates inaccurate outcomes.

Functional divergence of FimX in PilZ binding and type IV pilus regulation.

Type IV pili (T4P) are polar surface structures that play important roles in bacterial motility, biofilm formation, and pathogenicity. The protein FimX and its orthologs are known to mediate T4P formation in the human pathogen Pseudomonas aeruginosa and some other bacterial species. It was reported recently that FimX(XAC2398) from Xanthomonas axonopodis pv. citri interacts with PilZ(XAC1133) directly through the nonenzymatic EAL domain of FimX(XAC2398). Here we present experimental data to reveal that the strong interaction between FimX(XAC2398) and PilZ(XAC1133) is not conserved in P. aeruginosa and likely other Pseudomonas species. In vitro and in vivo binding experiments showed that the interaction between FimX and PilZ in P. aeruginosa is below the measurable limit. Surface plasmon resonance assays further confirmed that the interaction between the P. aeruginosa proteins is at least more than 3 orders of magnitude weaker than that between the X. axonopodis pv. citri pair. The N-terminal lobe region of FimX(XAC2398) was identified as the binding surface for PilZ(XAC1133) by amide hydrogen-deuterium exchange and site-directed mutagenesis studies. Lack of several key residues in the N-terminal lobe region of the EAL domain of FimX is likely to account for the greatly reduced binding affinity between FimX and PilZ in P. aeruginosa. All together, the results suggest that the interaction between PilZ and FimX in Xanthomonas species is not conserved in P. aeruginosa due to the evolutionary divergence among the FimX orthologs. The precise roles of FimX and PilZ in bacterial motility and T4P biogenesis are likely to vary among bacterial species.

Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change.

FimX is a multidomain signaling protein required for type IV pilus biogenesis and twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. FimX is localized to the single pole of the bacterial cell, and the unipolar localization is crucial for the correct assembly of type IV pili. FimX contains a non-catalytic EAL domain that lacks cyclic diguanylate (c-di-GMP) phosphodiesterase activity. It was shown that deletion of the EAL domain or mutation of the signature EVL motif affects the unipolar localization of FimX. However, it was not understood how the C-terminal EAL domain could influence protein localization considering that the localization sequence resides in the remote N-terminal region of the protein. Using hydrogen/deuterium exchange-coupled mass spectrometry, we found that the binding of c-di-GMP to the EAL domain triggers a long-range (∼ca. 70 Å) conformational change in the N-terminal REC domain and the adjacent linker. In conjunction with the observation that mutation of the EVL motif of the EAL domain abolishes the binding of c-di-GMP, the hydrogen/deuterium exchange results provide a molecular explanation for the mediation of protein localization and type IV pilus biogenesis by c-di-GMP through a remarkable allosteric regulation mechanism.

2',3'-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening.

The recent report of 2',3'-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2',3'-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2',3'-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3'-AMP but not 2'-AMP. The catalytic efficiency (k(cat)/K(m)) of each enzyme against 2',3'-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2',3'-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3'-AMP is due to the P-O2' bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2',3'-cAMP hydrolysis is observed.

A flavin cofactor-binding PAS domain regulates c-di-GMP synthesis in AxDGC2 from Acetobacter xylinum.

The cytoplasmic protein AxDGC2 regulates cellulose synthesis in the obligate aerobe Acetobacter xylinum by controlling the cellular concentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2 contains a Per-Arnt-Sim (PAS) domain and two putative catalytic domains (GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS domain of AxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently. The redox status of the FAD cofactor modulates the catalytic activity of the GGDEF domain for c-di-GMP synthesis, with the oxidized form exhibiting higher catalytic activity and stronger substrate inhibition. The results suggest that AxDGC2 is a signaling protein that regulates the cellular c-di-GMP level in response to the change in cellular redox status or oxygen concentration. Moreover, several residues predicated to be involved in FAD binding and signal transduction were mutated to examine the impact on redox potential and catalytic activity. Despite the minor perturbation of redox potential and unexpected modification of FAD in one of the mutants, none of the single mutations was able to completely disrupt the transmission of the signal to the GGDEF domain, indicating that the change in the FAD redox state can still trigger structural changes in the PAS domain probably by using substituted hydrogen-bonded water networks. Meanwhile, although the EAL domain of AxDGC2 was found to be catalytically inactive toward c-di-GMP, it was capable of hydrolyzing some phosphodiester bond-containing nonphysiological substrates. Together with the previously reported oxygen-dependent activity of the homologous AxPDEA1, the results provided new insight into relationships among oxygen level, c-di-GMP concentration, and cellulose synthesis in A. xylinum.

The functional role of a conserved loop in EAL domain-based cyclic di-GMP-specific phosphodiesterase.

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (beta/alpha)(8) barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K(m)) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K(m) correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg(2+) ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.

Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa.

EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second-messenger cyclic di-GMP in bacteria. Given the pivotal roles of EAL domains in the regulation of many bacterial behaviors, the elucidation of their catalytic and regulatory mechanisms would contribute to the effort of deciphering the cyclic di-GMP signaling network. Here, we present data to show that RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO-1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg(2+) ion. In addition to the five essential residues involved in Mg(2+) binding, we propose that the essential residue E(352) functions as a general base catalyst assisting the deprotonation of Mg(2+)-coordinated water to generate the nucleophilic hydroxide ion. The mutation of other conserved residues caused various degree of changes in the k(cat) or K(m), leading us to propose their roles in residue positioning and substrate binding. With functions assigned to the conserved groups in the active site, we discuss the molecular basis for the lack of activity of some characterized EAL domain proteins and the possibility of predicting the phosphodiesterase activities for the vast number of EAL domains in bacterial genomes in light of the catalytic mechanism.