Substrate ATP Research Articles

Hsp90, DnaK, and ClpB collaborate in protein reactivation

Hsp70, Hsp90, and ClpB/Hsp100 are molecular chaperones that help regulate proteostasis. Bacterial and yeast Hsp70s and their cochaperones function synergistically with Hsp90s to reactivate inactive and aggregated proteins by a mechanism that requires a direct interaction between Hsp90 and Hsp70 both in vitro and in vivo. Escherichia coli and yeast Hsp70s also collaborate in bichaperone systems with ClpB and Hsp104, respectively, to disaggregate and reactivate aggregated proteins and amyloids such as prions. These collaborations are dependent on direct interactions between ClpB/Hsp104 and Hsp70. We explored the possibility that E. coli homologs of Hsp70, Hsp90, and ClpB, referred to as DnaK, Hsp90Ec, and ClpB, respectively, in combination with two DnaK cochaperones, DnaJ and GrpE, could promote protein disaggregation and reactivation under conditions where bichaperone systems are ineffective. Our results show that Hsp90Ec is able to overcome the inhibition of protein disaggregation and reactivation observed when the concentration of DnaK is approaching physiological concentrations. We found that ATP hydrolysis and substrate binding by all three chaperones are essential for the collaborative function. The work further shows that ClpB acts early in protein reactivation with DnaK and its cochaperones; E. coli Hsp90 acts at a later stage after ClpB. The results highlight the collaboration among chaperones to regulate and maintain proteostasis.

Interplay between conformational dynamics and substrate binding regulates enzymatic activity: a single-molecule FRET study.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. We utilize this phenomenon to decipher the involvement of conformational dynamics in the mechanism of action of the enzyme. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Further, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results allow us to define a complete kinetic scheme that includes the open/close transitions of the enzyme and to unravel the important interplay between conformational dynamics and chemical steps, a general property of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.

ENPP1/CD203a-targeting heavy-chain antibody reveals cell-specific expression on human immune cells

ENPP1/CD203a is a membrane-bound ectonucleotidase capable of hydrolyzing ATP, cGAMP and other substrates. Its enzymatic activity plays an important role in the balance of extracellular adenine nucleotides and the modulation of purinergic signaling, in soft tissue calcification, and in the regulation of the cGAS/STING pathway. However, a detailed analysis of ENPP1 surface expression on human immune cells has not been performed. Here, we selected VHH domains from human ENPP1-immunized alpacas to generate heavy-chain antibodies targeting ENPP1, and analyzed cell surface expression on all circulating immune cell subsets using flow cytometry. We find high expression of ENPP1 in CD141high conventional dendritic cells (cDC1), while ENPP1 was not detectable on other dendritic cells and monocytes. In the lymphocytic compartment, only CD56bright natural killer cells and mucosal-associated invariant T cells (MAIT) express ENPP1. In contrast, all other T cell subpopulations, CD56dim natural killer cells and B lymphocytes do not or only minimally express ENPP1. In summary, we describe highly cell type-specific expression of ENPP1 in the immune system using a newly generated heavy-chain antibody. This reagent will help to decipher the function of ENPP1 in the regulation of the immune response, allow a quick identification of ENPP1-deficiency and of ENPP1-positive tumors, and constitutes the basis for targeted anti-tumor intervention.

Structural and biophysical characterization of the cytoplasmic domains of HprS kinase and its interactions with the cognate regulator HprR.

The HprSR constitutes the bacterial two-component regulatory system engaged by Escherichia coli to reduce the damaging effects of reactive chlorine and oxygen species present in its cytosol. Hypochlorous acid (HOCl) has been shown to be the molecule capable of activating of the HprSR system. HOCl is produced upon pathogen invasion by phagocytic cells of the human innate immune system, particularly neutrophils, to take advantage of its powerful antimicrobial attributes. Therefore, comprehensive studies concerning bacterial sensing and regulatory HprSR system are indispensable in understanding and effectively eliminating pathogens. Here we present the first crystal structure, solved at 1.7Å resolution, of the HprS cytoplasmic domains arranged as a homodimer. In both protomers, the catalytic ATP-binding domain contains a non-hydrolysable ATP analog coordinated by a magnesium ion. This structure allowed us to provide a detailed characterization of kinase-substrate interaction. Furthermore, the structural data are supported by biophysical studies of kinase interaction with cognate response regulator HprR and substrate ATP. The kinase activity is also assessed in the presence or absence of HprR.

β-hydroxybutyrate is a metabolic regulator of proteostasis in the aged and Alzheimer disease brain.

Loss of proteostasis is a hallmark of aging and Alzheimer disease (AD). We identify β-hydroxybutyrate (βHB), a ketone body, as a regulator of protein solubility. βHB primarily provides ATP substrate during periods of reduced glucose availability, and regulates other cellular processes through protein interactions. We demonstrate βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain invivo after delivery of a ketone ester. This mechanism is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity. We generate libraries of the βHB-induced protein insolublome using mass spectrometry proteomics, and identify common protein domains and upstream regulators. We show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain. These data indicate a metabolically regulated mechanism of proteostasis relevant to aging and AD.

Stochastic nature and physiological implications of 5'-NAD RNA cap in bacteria.

RNA 5'-modification with NAD+/NADH (oxidized/reduced nicotinamide adenine dinucleotide) has been found in bacteria, eukaryotes and viruses. 5'-NAD is incorporated into RNA by RNA polymerases (RNAPs) during the initiation of synthesis. It is unknown (i) which factors and physiological conditions permit substantial NAD incorporation into RNA in vivo and (ii) how 5'-NAD impacts gene expression and the fate of RNA in bacteria. Here we show in Escherichia coli that RNA NADylation is stimulated by low cellular concentration of the competing substrate ATP, and by weakening ATP contacts with RNAP active site. Additionally, RNA NADylation may be influenced by DNA supercoiling. RNA NADylation does not interfere with posttranscriptional RNA processing by major ribonuclease RNase E. It does not impact the base-pairing between RNAI, the repressor of plasmid replication, and its antisense target, RNAII. Leaderless NADylated model mRNA cI-lacZ is recognized by the 70S ribosome and is translated with the same efficiency as triphosphorylated cI-lacZ mRNA. Translation exposes the 5'-NAD of this mRNA to de-capping by NudC enzyme. We suggest that NADylated mRNAs are rapidly degraded, consistent with their low abundance in published datasets. Furthermore, we observed that ppGpp inhibits NudC de-capping activity, contributing to the growth phase-dependency of NADylated RNA levels.

Oxidative Stress Markers and Na,K-ATPase Enzyme Kinetics Are Altered in the Cerebellum of Zucker Diabetic Fatty fa/fa Rats: A Comparison with Lean fa/+ and Wistar Rats.

Type 2 diabetes mellitus has been referred to as being closely related to oxidative stress, which may affect brain functions and brain glucose metabolism due to its high metabolic activity and lipid-rich content. Na,K-ATPase is an essential enzyme maintaining intracellular homeostasis, with properties that can sensitively mirror various pathophysiological conditions such as diabetes. The goal of this study was to determine oxidative stress markers as well as Na,K-ATPase activities in the cerebellum of Zucker diabetic fatty (ZDF) rats depending on diabetes severity. The following groups of male rats were used: Wistar, ZDF Lean (fa/+), and ZDF (fa/fa) rats, arbitrarily divided according to glycemia into ZDF obese (ZO, less severe diabetes) and ZDF diabetic (ZOD, advanced diabetes) groups. In addition to basic biometry and biochemistry, oxidative stress markers were assessed in plasma and cerebellar tissues. The Na, K-ATPase enzyme activity was measured at varying ATP substrate concentrations. The results indicate significant differences in basic biometric and biochemical parameters within all the studied groups. Furthermore, oxidative damage was greater in the cerebellum of both ZDF (fa/fa) groups compared with the controls. Interestingly, Na,K-ATPase enzyme activity was highest to lowest in the following order: ZOD > ZO > Wistar > ZDF lean rats. In conclusion, an increase in systemic oxidative stress resulting from diabetic conditions has a significant impact on the cerebellar tissue independently of diabetes severity. The increased cerebellar Na,K-ATPase activity may reflect compensatory mechanisms in aged ZDF (fa/fa) animals, rather than indicating cerebellar neurodegeneration: a phenomenon that warrants further investigation.

Enzyme activation by urea reveals the interplay between conformational dynamics and substrate binding: a single-molecule FRET study.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. At high concentrations, AMP also operates as an allosteric inhibitor of the protein. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea interferes with two key mechanisms that contribute to enzyme efficacy. First, urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Second, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results signify the important interplay between conformational dynamics and chemical steps, including binding, in the activity of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.

Structural basis for substrate binding and selection by human mitochondrial RNA polymerase

The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and dNTPs. Closing of the fingers domain over the catalytic site results in delivery of both the templating DNA base and the substrate into the Insertion Site and recruitment of the catalytic magnesium ions. The cryo-EM data also reveal a conformation adopted by mtRNAP to reject a non-cognate substrate from its active site. Our findings establish a structural basis for substrate binding and suggest a unified mechanism of NTP selection for single-subunit RNAPs.

Structural Characterization of Heat Shock Protein 90β and Molecular Interactions with Geldanamycin and Ritonavir: A Computational Study.

Drug repositioning is an important therapeutic strategy for treating breast cancer. Hsp90β chaperone is an attractive target for inhibiting cell progression. Its structure has a disordered and flexible linker region between the N-terminal and central domains. Geldanamycin was the first Hsp90β inhibitor to interact specifically at the N-terminal site. Owing to the toxicity of geldanamycin, we investigated the repositioning of ritonavir as an Hsp90β inhibitor, taking advantage of its proven efficacy against cancer. In this study, we used molecular modeling techniques to analyze the contribution of the Hsp90β linker region to the flexibility and interaction between the ligands geldanamycin, ritonavir, and Hsp90β. Our findings indicate that the linker region is responsible for the fluctuation and overall protein motion without disturbing the interaction between the inhibitors and the N-terminus. We also found that ritonavir established similar interactions with the substrate ATP triphosphate, filling the same pharmacophore zone.

BsuMI regulates DNA transformation in Bacillus subtilis besides the defense system and the constructed strain with BsuMI-absence is applicable as a universal transformation platform for wild-type Bacillus

BackgroundTo effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process.ResultsTranscriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R−M− without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained.ConclusionsThe enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.Graphical

The Role of SF1 and SF2 Helicases in Biotechnological Applications.

Helicases, which utilize ATP hydrolysis to separate nucleic acid duplexes, play crucial roles in DNA and RNA replication, repair, recombination, and transcription. Categorized into the major groups superfamily 1 (SF1) and superfamily 2 (SF2), alongside four minor groups, these proteins exhibit a conserved catalytic core indicative of a shared evolutionary origin while displaying functional diversity through interactions with various substrates. This review summarizes the structures, functions and mechanisms of SF1 and SF2 helicases, with an emphasis on conserved ATPase sites and RecA-like domains essential for their enzymatic and nucleic acid binding capabilities. It highlights the unique 1B and 2B domains in SF1 helicases and their impact on enzymatic activity. The DNA unwinding process is detailed, covering substrate recognition, ATP hydrolysis, and conformational changes, while addressing debates over the active form of UvrD helicase and post-unwinding dissociation. More importantly, this review discusses the biotechnological potential of helicases in emerging technologies such as nanopore sequencing, protein sequencing, and isothermal amplification, focusing on their use in pathogen detection, biosensor enhancement, and cancer treatment. As understanding deepens, innovative applications in genome editing, DNA sequencing, and synthetic biology are anticipated.

Effects of substrate availability and mitochondrial disruption on oxidative metabolism and sperm motility in fertile dogs.

In several mammalian species, the measurement of mitochondrial oxygen consumption (MITOX) under different metabolic conditions has demonstrated a positive correlation with sperm motility and may be a sensitive indicator of mitochondrial health. In general, the maintenance of sperm motility and many key sperm functions and fertilizing events are heavily energy-dependent processes, and some species-specific substrate preferences exist. Although canine sperm have been known to undergo capacitation and maintain motility with supplementation of a wide range of energy substrates, the relationship between mitochondrial function, and the maintenance of oxidative metabolism and sperm motility remain unclear. The objective of this study was to explore the metabolic flexibility of canine sperm, and to investigate the relationship between mitochondrial function, and maintenance of motility under differing nutrient conditions. We explored substrate preferences and the bioenergetics underlying maintenance of canine sperm motility by monitoring mitochondrial oxidative function and sperm kinematics in the presence of mitochondrial effector drug treatments: FCCP, antimycin (ANTI), and oligomycin (OLIGO). We hypothesized that canine sperm possess the ability to use compensatory pathways and utilize diverse nutrient sources in the maintenance of motility. Oxygen consumption (change in pO2, oxygen partial pressure) and sperm kinematics (CASA) were measured concurrently (t0-t30) to assess the relationship between oxidative metabolism and maintenance of sperm motility in dogs. Four media were tested: containing glucose, lactate, and pyruvate (GLP), containing glucose (G), fructose (F), or lactate and pyruvate (LP). In the absence of pharmacological inhibition of the electron transport chain, energetic substrate had no effect on sperm kinematics in fertile dogs. Following mitochondrial disruption by ANTI and OLIGO, mitochondrial oxygen consumption was negatively correlated with several sperm motility parameters in GLP, G, F, and LP media. In every media, FCCP treatment quickly induced significantly higher oxygen consumption than in untreated sperm, and spare respiratory capacity, the maximal inducible oxidative metabolism, was high. With respiratory control ratios RCR >1 there was no indication of bioenergetic dysfunction in any media type, indicating that sperm mitochondria of fertile dogs have a high capacity for substrate oxidation and ATP turnover regardless of substrate. Our results suggest MITOX assessment is a valuable tool for assessing mitochondrial functionality, and that canine sperm employ flexible energy management systems which may be exploited to improve sperm handling and storage.

Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes

The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5′-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2–8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2–8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4′-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.

Abstract 2472: Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models

Abstract Background: ENPP1, a nucleotide pyrophosphatase and phosphodiesterase, has emerged as an important modulator of anti-tumor immune activation, as it regulates the concentrations of cGAMP and ATP in the TME. ENPP1 expression in tumors is common and correlates with a cold tumor phenotype. Its loss has been reported to suppress metastasis, restore immune cell infiltration into the tumor and potentiate the response to immune checkpoint blockade, while, conversely, the overexpression of ENPP1 promoted metastasis and rendered tumors resistant to checkpoint blockade. RBS2418 was designed as a potent and selective inhibitor of ENPP1 and is in clinical development. Methods: ENPP1 enzyme inhibition was measured in vitro using cGAMP and ATP substrates. Effects of pH and serum and selectivity against related enzymes were evaluated. In vivo efficacy was assessed in Hepa1-6, and GL261-luc syngeneic liver and glioblastoma mouse models. Compound exposures were determined in tumor and blood samples. Cures were confirmed by re-challenge of cured animals with the same cancer cells. Results: RBS2418 inhibited hydrolysis of cGAMP and ATP in vitro with similar potency (Ki =0.14 and 0.13 nM). Inhibition was also similar in human serum, consistent with low binding affinity to human serum albumin. In humans, oral dosing at doses of 100 mg could inhibit ENPP1 enzyme activity during the full dosing interval. Dosing of RBS2418 was performed in mice to achieve trough concentrations of RBS2418 in tumors and plasma exceeding ENPP1 EC90 levels for the complete dosing interval. In the Hepa1-6 model treatment with RBS2418 for either 2 or 10 days resulted in significant reduction of tumor growth and prolongation of survival as compared to vehicle control. There was no significant difference between the RBS2418 groups. Complete tumor regression was observed in 44% of treated mice across the two treatment groups. These mice were resistant to rechallenge with Hepa1-6 tumor cells. In the GL261-luc model treatment with RBS2418 showed significant reduction of tumor burden and prolongation of survival as compared to vehicle control. The mice that achieved complete tumor regression were resistant to rechallenge with GL261-luc tumor cells. Conclusions: RBS2418 inhibited ATP and cGAMP hydrolysis by ENPP1 with similar high potency. The pharmacology profile was optimized to achieve low serum protein binding and oral bioavailability in humans. RBS2418 treatment was well tolerated in mice under conditions where tumor and plasma levels exceeding ENPP1 serum EC90 levels were achieved. In the syngeneic mouse models Hepa1-6 and GL261-luc significant tumor growth reductions, complete tumor regression and cures were observed. Resistance to cancer cell rechallenge was consistent with treatment-induced anti-tumor immunity. Citation Format: Ningwu Huang, Junjun Cheng, Ron Hawley, Klaus Klumpp. Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2472.

Discovering potential WRN inhibitors from natural product database through computational methods

Microsatellite instability (MSI) is a relatively common feature associated with multiple cancers, and Werner syndrome (WRN) ATP-dependent helicase has been recognized as a novel target for treating MSI cancers, such as colorectal cancer. A small-molecule inhibitor targeting WRN would be a promising strategy for treating colorectal cancer with high MSI expression. In this study, we employed a computer-assisted drug discovery strategy to screen over 30,000 natural product molecules. By using a combination of docking, ligand efficiency, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), and thermodynamic integration (TI) calculations, we identified MOL008980, MOL010740, MOL011832, T4743, TN1166, and TNP-002173 as potential WRN inhibitors. Subsequent molecular dynamics simulation revealed that these screened natural products possessed better binding dynamic characteristics than ATP substrate and were capable of inhibiting the dynamic process of WRN, making them potential strong ATP competitive inhibitors. In conclusion, our computational approach revealed potential WRN inhibitors from a natural product database, providing a theoretical basis for future research.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

Ovarian cancer cells regulate their mitochondrial content and high mitochondrial content is associated with a poor prognosis

Most cancer patients ultimately die from the consequences of distant metastases. As metastasis formation consumes energy mitochondria play an important role during this process as they are the most important cellular organelle to synthesise the energy rich substrate ATP, which provides the necessary energy to enable distant metastasis formation. However, mitochondria are also important for the execution of apoptosis, a process which limits metastasis formation. We therefore wanted to investigate the mitochondrial content in ovarian cancer cells and link its presence to the patient’s prognosis in order to analyse which of the two opposing functions of mitochondria dominates during the malignant progression of ovarian cancer. Monoclonal antibodies directed against different mitochondrial specific proteins, namely heat shock proteins 60 (HSP60), fumarase and succinic dehydrogenase, were used in immunohistochemistry in preliminary experiments to identify the antibody most suited to detect mitochondria in ovarian cancer cells in clinical tissue samples. The clearest staining pattern, which even delineated individual mitochondria, was seen with the anti-HSP60 antibody, which was used for the subsequent clinical study staining primary ovarian cancers (n = 155), borderline tumours (n = 24) and recurrent ovarian cancers (n = 26). The staining results were semi-quantitatively scored into three groups according to their mitochondrial content: low (n = 26), intermediate (n = 50) and high (n = 84). Survival analysis showed that high mitochondrial content correlated with a statistically significant overall reduced survival rate In addition to the clinical tissue samples, mitochondrial content was analysed in ovarian cancer cells grown in vitro (cell lines: OVCAR8, SKOV3, OVCAR3 and COV644) and in vivo in severe combined immunodeficiency (SCID) mice.In in vivo grown SKOV3 and OVCAR8 cells, the number of mitochondria positive cells was markedly down-regulated compared to the in vitro grown cells indicating that mitochondrial number is subject to regulatory processes. As high mitochondrial content is associated with a poor prognosis, the provision of high energy substrates by the mitochondria seems to be more important for metastasis formation than the inhibition of apoptotic cell death, which is also mediated by mitochondria. In vivo and in vitro grown human ovarian cancer cells showed that the mitochondrial content is highly adaptable to the growth condition of the cancer cells.

Identification of FtfL as a novel target of berberine in intestinal bacteria

BackgroundBerberine (BBR) is a commonly used anti-intestinal inflammation drug, and its anti-cancer activity has been found recently. BBR can intervene and control malignant colorectal cancer (CRC) through intestinal microbes, but the direct molecular target and related mechanism are unclear. This study aimed to identify the target of BBR and dissect related mechanisms against the occurrence and development of CRC from the perspective of intestinal microorganisms.ResultsHere, we found that BBR inhibits the growth of several CRC-driving bacteria, especially Peptostreptococcus anaerobius. By using a biotin-conjugated BBR derivative, we identified the protein FtfL (formate tetrahydrofolate ligase), a key enzyme in C1 metabolism, is the molecular target of BBR in P. anaerobius. BBR exhibits strong binding affinity and potent inhibition on FtfL. Based on this, we determined the crystal structure of PaFtfL (P. anaerobius FtfL)-BBR complex and found that BBR can not only interfere with the conformational flexibility of PaFtfL tetramer by wedging the tetramer interface but also compete with its substrate ATP for binding within the active center. In addition, the enzymatic activities of FtfL homologous proteins in human tumor cells can also be inhibited by BBR.ConclusionsIn summary, our study has identified FtfL as a direct target of BBR and uncovered molecular mechanisms involved in the anti-CRC of BBR. BBR interferes with intestinal pathogenic bacteria by targeting FtfLs, suggesting a new means for controlling the occurrence and development of CRC.

Selenite ameliorates the ATP hydrolysis of mitochondrial F1FO-ATPase by changing the redox state of thiol groups and impairs the ADP phosphorylation

Selenite as an inorganic form of selenium can affect the redox state of mitochondria by modifying the thiol groups of cysteines. The F1FO-ATPase has been identified as a mitochondrial target of this compound. Indeed, the bifunctional mechanism of ATP turnover of F1FO-ATPase was differently modified by selenite. The activity of ATP hydrolysis was stimulated, whereas the ADP phosphorylation was inhibited. We ascertain that a possible new protein adduct identified as seleno-dithiol (-S-Se-S-) mercaptoethanol-sensitive caused the activation of F-ATPase activity and the oxidation of free –SH groups in mitochondria. Conversely, the inhibition of ATP synthesis by selenite might be irreversible. The kinetic analysis of the activation mechanism was an uncompetitive mixed type with respect to the ATP substrate. Selenite bound more selectively to the F1FO-ATPase loaded with the substrate by preferentially forming a tertiary (enzyme-ATP-selenite) complex. Otherwise, the selenite was a competitive mixed-type activator with respect to the Mg2+ cofactor. Thus, selenite more specifically bound to the free enzyme forming the complex enzyme-selenite. However, even if the selenite impaired the catalysis of F1FO-ATPase, the mitochondrial permeability transition pore phenomenon was unaffected. Therefore, the reversible energy transduction mechanism of F1FO-ATPase can be oppositely regulated by selenite.