Structural Insights into Protein-Inhibitor Interactions in Human Tryptophan Dioxygenase.

Human tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two important targets in cancer immunotherapy. Extensive research has led to a large number of potent IDO inhibitors; in addition, 52 structures of IDO in complex with inhibitors with a wide array of chemical scaffolds have been documented. In contrast, progress in the development of TDO inhibitors has been limited. Only four structures of TDO in complex with competitive inhibitors that compete with the substrate L-tryptophan for binding to the active site have been reported to date. Here we systematically evaluated the structures of TDO in complex with competitive inhibitors with three types of pharmacophores, imidazo-isoindole, indole-tetrazole, and indole-benzotriazole. The comparative assessment of the protein-inhibitor interactions sheds new light into the structure-based design of enzyme-selective inhibitors.

Advancements in inhibitors of crucial enzymes in the cysteine biosynthetic pathway: Serine acetyltransferase and O-acetylserine sulfhydrylase.

Infectious diseases have been jeopardized problem that threaten public health over a long period of time. The growing prevalence of drug-resistant pathogens and infectious cases have led to a decrease in the number of effective antibiotics, which highlights the urgent need for the development of new antibacterial agents. Serine acetyltransferase (SAT), also known as CysE in certain bacterial species, and O-acetylserine sulfhydrylase (OASS), also known as CysK in select bacteria, are indispensable enzymes within the cysteine biosynthesis pathway of various pathogenic microorganisms. These enzymes play a crucial role in the survival of these pathogens, making SAT and OASS promising targets for the development of novel anti-infective agents. In this comprehensive review, we present an introduction to the structure and function of SAT and OASS, along with an overview of existing inhibitors for SAT and OASS as potential antibacterial agents. Our primary focus is on elucidating the inhibitory activities, structure-activity relationships, and mechanisms of action of these inhibitors. Through this exploration, we aim to provide insights into promising strategies and prospects in the development of antibacterial agents that target these essential enzymes.

Bridging in silico and in vitro perspectives to unravel molecular mechanisms underlying the inhibition of ß-glucuronidase by coumarins from Hibiscus trionum.

Unraveling the intricacies of ß-glucuronidase inhibition is pivotal for developing effective strategies in applications specific to gastrointestinal health and drug metabolism. Our study investigated the efficacy of some Hibiscus trionum phytochemicals as ß-glucuronidase inhibitors. The results showed that cleomiscosin A and mansonone H emerged as the most potent inhibitors, with IC50 values of 3.97 ± 0.35 µM and 10.32 ± 1.85 µM, respectively. Mechanistic analysis of ß-glucuronidase inhibition indicated that cleomiscosin A and the reference drug EGCG displayed a mixed inhibition mode against ß-glucuronidase, while mansonone H exhibited noncompetitive inhibition against ß-glucuronidase. Docking studies revealed that cleomiscosin A and mansonone H exhibited the lowest binding affinities, occupying the same site as EGCG, and engaged significant key residues in their binding mechanisms. Using a 30 ns molecular dynamics (MD) simulation, we explored the interaction dynamics of isolated compounds with ß-glucuronidase. Analysis of various MD parameters showed that cleomiscosin A and mansonone H exhibited consistent trajectories and significant energy stabilization with ß-glucuronidase. These computational insights complemented experimental findings, underscoring the potential of cleomiscosin A and mansonone H as ß-glucuronidase inhibitors.

Mining the Dynamical Properties of Substrate and FAD Binding Pockets of LSD1: Hints for New Inhibitor Design Direction.

Lysine-specific demethylase 1 (LSD1), a highly sophisticated epigenetic regulator, orchestrates a range of critical cellular processes, holding promising therapeutic potential for treating diverse diseases. However, the clinical research progress targeting LSD1 is very slow. After 20 years of research, only one small-molecule drug, BEA-17, targeting the degradation of LSD1 and CoREST has been approved by the U.S. Food and Drug Administration. The primary reason for this may be the lack of abundant structural data regarding its intricate functions. To gain a deeper understanding of its conformational dynamics and guide the drug design process, we conducted molecular dynamics simulations to explore the conformational states of LSD1 in the apo state and under the influence of cofactors of flavin adenine dinucleotide (FAD) and CoREST. Our results showed that, across all states, the substrate binding pocket exhibited high flexibility, whereas the FAD binding pocket remained more stable. These distinct dynamical properties are essential for LSD1's ability to bind various substrates while maintaining efficient demethylation activity. Both pockets can be enlarged by merging with adjacent pockets, although only the substrate binding pocket can shrink into smaller pockets. These new pocket shapes can inform inhibitor design, particularly for selectively FAD-competitive inhibitors of LSD1, given the presence of numerous FAD-dependent enzymes in the human body. More interestingly, in the absence of FAD binding, the united substrate and FAD binding pocket are partitioned by the conserved residue of Tyr761, offering valuable insights for the design of inhibitors that disrupt the crucial steric role of Tyr761 and the redox role of FAD. Additionally, we identified pockets that positively or negatively correlate with the substrate and FAD binding pockets, which can be exploited for the design of allosteric or concurrent inhibitors. Our results reveal the intricate dynamical properties of LSD1 as well as multiple novel conformational states, which deepen our understanding of its sophisticated functions and aid in the rational design of new inhibitors.

An expanded trove of fragment-bound structures for the allosteric enzyme PTP1B from computational reanalysis of large-scale crystallographic data.

Due to their low binding affinities, detecting small-molecule fragments bound to protein structures from crystallographic datasets has been a challenge. Here, we report a trove of 65 new fragment hits for PTP1B, an "undruggable" therapeutic target enzyme for diabetes and cancer. These structures were obtained from computational analysis of data from a large crystallographic screen, demonstrating the power of this approach to elucidate many (∼50% more) "hidden" ligand-bound states of proteins. Our new structures include a fragment hit found in a novel binding site in PTP1B with a unique location relative to the active site, one that links adjacent allosteric sites, and, perhaps most strikingly, a fragment that induces long-range allosteric protein conformational responses. Altogether, our research highlights the utility of computational analysis of crystallographic data, makes publicly available dozens of new ligand-bound structures of a high-value drug target, and identifies novel aspects of ligandability and allostery in PTP1B.

SHP2-EGFR States in Dephosphorylation Can Inform Selective SHP2 Inhibitors, Dampening RasGAP Action.

SHP2 is a positive regulator of the EGFR-dependent Ras/MAPK pathway. It dephosphorylates a regulatory phosphorylation site in EGFR that serves as the binding site to RasGAP (RASA1 or p120RasGAP). RASA1 is activated by binding to the EGFR phosphate group. Active RASA1 deactivates Ras by hydrolyzing Ras-bound GTP to GDP. Thus, SHP2 dephosphorylation of EGFR effectively prevents RASA1-mediated deactivation of Ras, thereby stimulating proliferation. Despite knowledge of this vital regulation in cell life, mechanistic in-depth structural understanding of the involvement of SHP2, EGFR, and RASA1 in the Ras/MAPK pathway has largely remained elusive. Here we elucidate the interactions, the factors influencing EGFR's recruitment of RASA1, and SHP2's recognition of the substrate site in EGFR. We reveal that RASA1 specifically interacts with the DEpY992LIP motif in EGFR featuring a proline residue at the +3 position C-terminal to pY primarily through its nSH2 domain. This interaction is strengthened by the robust attraction of two acidic residues, E991 and D990, of EGFR to two basic residues in the BC-loop near the pY-binding pocket of RASA1's nSH2. In the stable precatalytic state of SHP2 with EGFR (DADEpY992LIPQ), the E-loop of SHP2's active site favors the interaction with the (-2)-position D990 and (-4)-position D988 N-terminal to pY992 in EGFR, while the pY-loop constrains the (+4)-position Q996 C-terminal to pY992. These specific interactions not only provide a structural basis for identifying negative regulatory sites in other RTKs but can inform selective, high-affinity active-site SHP2 inhibitors tailored for SHP2 mutants.

Isolation, Characterization, Genome Annotation, and Evaluation of Hyaluronidase Inhibitory Activity in Secondary Metabolites of Brevibacillus sp. JNUCC 41: A Comprehensive Analysis through Molecular Docking and Molecular Dynamics Simulation.

Brevibacillus sp. JNUCC 41, characterized as a plant-growth-promoting rhizobacterium (PGPR), actively participates in lipid metabolism and biocontrol based on gene analysis. This study aimed to investigate the crucial secondary metabolites in biological metabolism; fermentation, extraction, and isolation were performed, revealing that methyl indole-3-acetate showed the best hyaluronidase (HAase) inhibitory activity (IC50: 343.9 µM). Molecular docking results further revealed that the compound forms hydrogen bonds with the residues Tyr-75 and Tyr-247 of HAase (binding energy: -6.4 kcal/mol). Molecular dynamics (MD) simulations demonstrated that the compound predominantly binds to HAase via hydrogen bonding (MM-PBSA binding energy: -24.9 kcal/mol) and exhibits good stability. The residues Tyr-247 and Tyr-202, pivotal for binding in docking, were also confirmed via MD simulations. This study suggests that methyl indole-3-acetate holds potential applications in anti-inflammatory and anti-aging treatments.

Characterization of the Bottlenecks and Pathways for Inhibitor Dissociation from [NiFe] Hydrogenase.

[NiFe] hydrogenases can act as efficient catalysts for hydrogen oxidation and biofuel production. However, some [NiFe] hydrogenases are inhibited by gas molecules present in the environment, such as O2 and CO. One strategy to engineer [NiFe] hydrogenases and achieve O2- and CO-tolerant enzymes is by introducing point mutations to block the access of inhibitors to the catalytic site. In this work, we characterized the unbinding pathways of CO in the complex with the wild-type and 10 different mutants of [NiFe] hydrogenase from Desulfovibrio fructosovorans using τ-random accelerated molecular dynamics (τRAMD) to enhance the sampling of unbinding events. The ranking provided by the relative residence times computed with τRAMD is in agreement with experiments. Extensive data analysis of the simulations revealed that from the two bottlenecks proposed in previous studies for the transit of gas molecules (residues 74 and 122 and residues 74 and 476), only one of them (residues 74 and 122) effectively modulates diffusion and residence times for CO. We also computed pathway probabilities for the unbinding of CO, O2, and H2 from the wild-type [NiFe] hydrogenase, and we observed that while the most probable pathways are the same, the secondary pathways are different. We propose that introducing mutations to block the most probable paths, in combination with mutations to open the main secondary path used by H2, can be a feasible strategy to achieve CO and O2 resistance in the [NiFe] hydrogenase from Desulfovibrio fructosovorans.

Antioxidant capacity of xylooligosaccharides generated from beechwood xylan by recombinant family GH10 Aspergillus niger xylanase A and insights into the enzyme's competitive inhibition by riceXIP.

In this study, the family GH10 xylanase AnXylA10 derived from Aspergillus niger JL15 strain was expressed in Pichia pastoris X33. The recombinant xylanase, reAnXylA10 exhibited optimal activity at 40 ℃ and pH 5.0. The hydrolysates generated from beechwood xylan using reAnXylA10 primarily consisted of xylobiose (X2) to xylohexaose (X6) and demonstrated remarkable antioxidant capacity. Furthermore, the rice xylanase inhibitory protein (riceXIP) was observed to competitively inhibit reAnXylA10, exhibiting an inhibition constant (Ki) of 140.6 nM. Molecular dynamics (MD) simulations of AnXylA10-riceXIP complex revealed that the α-7 helix (Q225-S238) of riceXIP intruded into the catalytic pocket of AnXylA10, thereby obstructing substrate access to the active site. Specifically, residue K226 of riceXIP formed robust interactions with E136 and E242, the two catalytic sites of AnXylA10, predominantly through high-occupied hydrogen bonds. Based on QTAIM, electron densities for the atom pairs K226riceXIP@HZ1-E136AnXylA10@OE2 and K226riceXIP@HZ3-E242AnXylA10@OE1 were determined to be 0.04628 and 0.02914 a.u., respectively. Binding free energy of AnXylA10-riceXIP complex was -59.0±7.6 kcal/mol, significantly driven by electrostatic and van der Waals forces. Gaining insights into the interaction between xylanase and its inhibitors, and mining the inhibition mechanism in depth, will facilitate the design of innovative GH10 family xylanases that are both highly efficient and resistant to inhibitors.

Interactions of Phenylalanine Derivatives with Human Tyrosinase: Lessons from Experimental and Theoretical tudies.

The pigmentation of the skin, modulated by different actors in melanogenesis, is mainly due to the melanins (protective pigments). In humans, these pigments' precursors are synthetized by an enzyme known as tyrosinase (TyH). The regulation of the enzyme activity by specific modulators (inhibitors or activators) can offer a means to fight hypo- and hyper-pigmentations responsible for medical, psychological and societal handicaps. Herein, we report the investigation of phenylalanine derivatives as TyH modulators. Interacting with the binuclear copper active site of the enzyme, phenylalanine derivatives combine effects induced by combination with known resorcinol inhibitors and natural substrate/intermediate (amino acid part). Computational studies including docking, molecular dynamics and free energy calculations combined with biological activity assays on isolated TyH and in human melanoma MNT-1 cells, and X-ray crystallography analyses with the TyH analogue Tyrp1, provide conclusive evidence of the interactions of phenylalanine derivatives with human tyrosinase. In particular, our findings indicate that an analogue of L-DOPA, namely (S)-3-amino-tyrosine, stands out as an amino phenol derivative with inhibitory properties against TyH.

Telomerase inhibitors induce mitochondrial oxidation and DNA damage-dependent cell death rescued by Bcl-2/Bcl-xL.

BACKGROUND: Reactivation of telomerase is a hallmark of cancer and the majority of cancers over-express telomerase. Telomerase-dependent telomere length maintenance confers immortality to cancer cells. However, telomere length-independent cell survival functions of telomerase also play a critical role in tumorigenesis. Multiple telomerase inhibitors have been developed as therapeutics and include anti-sense oligonucleotides, telomerase RNA component targeting agents, chemical inhibitors of telomerase, small molecule inhibitors of hTERT, and telomerase vaccine. In general, telomerase inhibitors affect cell proliferation and survival of cells depending on the telomere length reduction, culminating in replicative senescence or cell death by crisis. However, most telomerase inhibitors kill cancer cells prior to significant reduction in telomere length, suggesting telomere length independent role of telomerase in early telomere dysfunction-dependent cell death. METHODS: In this study, we explored the mechanism of cell death induced by three prominent telomerase inhibitors utilizing a series of genetically encoded sensor cells including redox and DNA damage sensor cells. RESULTS: We report that telomerase inhibitors induce early cell cycle inhibition, followed by redox alterations at cytosol and mitochondria. Massive mitochondrial oxidation and DNA damage induce classical cell death involving mitochondrial transmembrane potential loss and mitochondrial permeabilization. Real-time imaging of the progression of mitochondrial oxidation revealed that treated cells undergo a biphasic mitochondrial redox alteration during telomerase inhibition, emphasizing the potential role of telomerase in the redox regulation at mitochondria. Additionally, silencing of hTERT confirmed its predominant role in maintaining mitochondrial redox homeostasis. Interestingly, the study also demonstrated that anti-apoptotic Bcl-2 family proteins still confer protection against cell death induced by telomerase inhibitors. CONCLUSION: The study demonstrates that redox alterations and DNA damage contribute to early cell death by telomerase inhibitors and anti-apoptotic Bcl-2 family proteins confer protection from cell death by their ability to safeguard mitochondria from oxidation damage.

Structural and functional characterization of mycobacterial PhoH2 and identification of potential inhibitor of its enzymatic activity.

Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, we elaborated the characterization of Mt-PhoH2 through biophysical, biochemical, and computational methods. In addition to its intrinsic ATPase activity, the biochemical experiments revealed its GTPase activity and both activities are metal ion dependent. Magnesium, manganese, copper, iron, nickel, zinc, cesium, calcium, and lithium were examined for their effect on activity, and the optimum activity was found with 10 mM of Mg2+ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as Km 4.873 ± 0.44 µM, Vmax 12.3817 ± 0.084 µM/min/mg, Kcat 0.0075 ± 0.00005 s-1, and Kcat/Km 0.0015 ± 0.000001 µM-1 s-1 with GTP. In the case of GTP as a substrate, a 20% decrease in enzymatic activity and a 50% increase in binding affinity of Mt-PhoH2 were observed. The substrates ADP and GDP inhibit the ATPase and GTPase activity of Mt-PhoH2. CD spectroscopy showed the dominance of alpha helix in the secondary structure of Mt-PhoH2, and this structural pattern was altered upon addition of ATP and GTP. In silico inhibitor screening revealed ML141 and NAV_2729 as two potential inhibitors of the catalytic activity of Mt-PhoH2. Mt-PhoH2 is essential for mycobacterial growth as its knockdown strain showed a decreased growth effect. Overall, the present article emphasizes the factors essential for the proper functioning of Mt-PhoH2 which is a participant in the toxin-antitoxin machinery and may also play an important role in phosphate starvation.

Structure-based discovery of a new series of nucleoside-derived ring-opening PRMT5 inhibitors.

The ubiquitous methyltransferases employing SAM as the methyl donor have emerged as potential targets in many disease treatments, especially in anticancer. Therefore, developing SAM-competitive inhibitors of methyltransferases is of great interest to the drug research. To explore this direction, herein, we rationally designed a series of nucleoside derivatives as potent PRMT5 inhibitors with novel scaffold. The representative compounds A2 and A8 exhibited highly potent PRMT5 inhibition activity as well as good selectivity over other PRMTs and PKMTs. Further cellular experiments revealed that compounds A2 and A8 potently reduced the level of sDMA and inhibited the proliferation of Z-138 and MOLM-13 cell lines by inducing apoptosis. Moreover, compounds A8 which had favorable pharmacokinetic properties exhibited potent antitumor efficacy without the loss of body weight in a subcutaneous MOLM-13 xenograft model. In summary, our efforts provided a series of novel nucleoside analogs as potent PRMT5 inhibitors and may also offer a new strategy to develop SAM analogs as other methyltransferases' inhibitors.

Discovery of Nanomolar Inhibitors for Human Dihydroorotate Dehydrogenase Using Structure-Based Drug Discovery Methods.

We used a structure-based drug discovery approach to identify novel inhibitors of human dihydroorotate dehydrogenase (DHODH), which is a therapeutic target for treating cancer and autoimmune and inflammatory diseases. In the case of acute myeloid leukemia, no previously discovered DHODH inhibitors have yet succeeded in this clinical application. Thus, there remains a strong need for new inhibitors that could be used as alternatives to the current standard-of-care. Our goal was to identify novel inhibitors of DHODH. We implemented prefiltering steps to omit PAINS and Lipinski violators at the earliest stages of this project. This enriched compounds in the data set that had a higher potential of favorable oral druggability. Guided by Glide SP docking scores, we found 20 structurally unique compounds from the ChemBridge EXPRESS-pick library that inhibited DHODH with IC50, DHODH values between 91 nM and 2.7 µM. Ten of these compounds reduced MOLM-13 cell viability with IC50, MOLM-13 values between 2.3 and 50.6 µM. Compound 16 (IC50, DHODH = 91 nM) inhibited DHODH more potently than the known DHODH inhibitor, teriflunomide (IC50, DHODH = 130 nM), during biochemical characterizations and presented a promising scaffold for future hit-to-lead optimization efforts. Compound 17 (IC50, MOLM-13 = 2.3 µM) was most successful at reducing survival in MOLM-13 cell lines compared with our other hits. The discovered compounds represent excellent starting points for the development and optimization of novel DHODH inhibitors.

High-resolution double vision of the allosteric phosphatase PTP1B.

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in cellular homeostasis and is a highly validated therapeutic target for multiple human ailments, including diabetes, obesity and breast cancer. However, much remains to be learned about how conformational changes may convey information through the structure of PTP1B to enable allosteric regulation by ligands or functional responses to mutations. High-resolution X-ray crystallography can offer unique windows into protein conformational ensembles, but comparison of even high-resolution structures is often complicated by differences between data sets, including non-isomorphism. Here, the highest resolution crystal structure of apo wild-type (WT) PTP1B to date is presented out of a total of ∼350 PTP1B structures in the PDB. This structure is in a crystal form that is rare for PTP1B, with two unique copies of the protein that exhibit distinct patterns of conformational heterogeneity, allowing a controlled comparison of local disorder across the two chains within the same asymmetric unit. The conformational differences between these chains are interrogated in the apo structure and between several recently reported high-resolution ligand-bound structures. Electron-density maps in a high-resolution structure of a recently reported activating double mutant are also examined, and unmodeled alternate conformations in the mutant structure are discovered that coincide with regions of enhanced conformational heterogeneity in the new WT structure. These results validate the notion that these mutations operate by enhancing local dynamics, and suggest a latent susceptibility to such changes in the WT enzyme. Together, these new data and analysis provide a detailed view of the conformational ensemble of PTP1B and highlight the utility of high-resolution crystallography for elucidating conformational heterogeneity with potential relevance for function.

PP2A inhibitor SET promotes mTORC1 and Bmi1 signaling through Akt activation and maintains the colony-formation ability of cancer cells.

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.

Anaerobic biodegradation of benzo(a)pyrene by a novel Cellulosimicrobium cellulans CWS2 isolated from polycyclic aromatic hydrocarbon-contaminated soil

Abstract Cellulosimicrobium cellulans CWS2, a novel strain capable of utilizing benzo(a)pyrene (BaP) as the sole carbon and energy source under nitrate-reducing conditions, was isolated from PAH-contaminated soil. Temperature and pH significantly affected BaP biodegradation, and the strain exhibited enhanced biodegradation ability at temperatures above 30 °C and between pH 7 and 10. The highest BaP removal rate (78.8%) was observed in 13 days when the initial BaP concentration was 10 mg/L, and the strain degraded BaP at constant rate even at a higher concentration (50 mg/L). Metal exposure experimental results illustrated that Cd(II) was the only metal ion that significantly inhibited biodegradation of BaP. The addition of 0.5 and 1.0 g/L glucose enhanced BaP biodegradation, while the addition of low-molecular-weight organic acids with stronger acidity reduced BaP removal rates during co-metabolic biodegradation. The addition of phenanthrene and pyrene, which were degraded to some extent by the strain, showed no distinct effect on BaP biodegradation. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the five rings of BaP opened, producing compounds with one to four rings which were more bioavailable. Thus, the strain exhibited strong BaP degradation capability and has great potential in the remediation of BaP-/PAH-contaminated environments.

Exenatide ameliorates hepatic steatosis and attenuates fat mass and FTO gene expression through PI3K signaling pathway in nonalcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a common disease associated with metabolic syndrome and can lead to life-threatening complications like hepatic carcinoma and cirrhosis. Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist antidiabetic drug, has the capacity to overcome insulin resistance and attenuate hepatic steatosis but the specific underlying mechanism is unclear. This study was designed to investigate the underlying molecular mechanisms of exenatide therapy on NAFLD. We used in vivo and in vitro techniques to investigate the protective effects of exenatide on fatty liver via fat mass and obesity associated gene (FTO) in a high-fat (HF) diet-induced NAFLD animal model and related cell culture model. Exenatide significantly decreased body weight, serum glucose, insulin, insulin resistance, serum free fatty acid, triglyceride, total cholesterol, low-density lipoprotein, aspartate aminotransferase, and alanine aminotransferase levels in HF-induced obese rabbits. Histological analysis showed that exenatide significantly reversed HF-induced lipid accumulation and inflammatory changes accompanied by decreased FTO mRNA and protein expression, which were abrogated by PI3K inhibitor LY294002. This study indicated that pharmacological interventions with GLP-1 may represent a promising therapeutic strategy for NAFLD.

Can microcystins affect zooplankton structure community in tropical eutrophic reservoirs? / Microcistinas podem afetar a estrutura da comunidade zooplanctônica em reservatórios eutróficos tropicais?

Abstract The aim of our study was to assess whether cyanotoxins (microcystins) can affect the composition of the zooplankton community, leading to domination of microzooplankton forms (protozoans and rotifers). Temporal variations in concentrations of microcystins and zooplankton biomass were analyzed in three eutrophic reservoirs in the semi-arid northeast region of Brazil. The concentration of microcystins in water proved to be correlated with the cyanobacterial biovolume, indicating the contributions from colonial forms such as Microcystis in the production of cyanotoxins. At the community level, the total biomass of zooplankton was not correlated with the concentration of microcystin (r2 = 0.00; P > 0.001), but in a population-level analysis, the biomass of rotifers and cladocerans showed a weak positive correlation. Cyclopoid copepods, which are considered to be relatively inefficient in ingesting cyanobacteria, were negatively correlated (r2 = – 0.01; P > 0.01) with the concentration of cyanotoxins. Surprisingly, the biomass of calanoid copepods was positively correlated with the microcystin concentration (r2 = 0.44; P > 0.001). The results indicate that allelopathic control mechanisms (negative effects of microcystin on zooplankton biomass) do not seem to substantially affect the composition of mesozooplankton, which showed a constant and high biomass compared to the microzooplankton (rotifers). These results may be important to better understand the trophic interactions between zooplankton and cyanobacteria and the potential effects of allelopathic compounds on zooplankton.

Resumo Com o objetivo de avaliar se as cianotoxinas (microcistinas) podem afetar a composição da comunidade zooplanctônica, levando à dominância de formas microzooplanctônicas (protozoários e rotiferos), as variações nas concentrações de microcistina e a biomassa do zooplâncton foram analisadas em três reservatórios eutróficos na região semi-árida do nordeste brasileiro. A concentração de microcistinas na água esteve correlacionada com o biovolume de cianobactérias, indicando a contribuição de formas coloniais como Microcystis na produção de cianotoxinas. A nível de comunidade, a biomassa total do zooplâncton não apresentou correlacão com a concentração de microcistina (r2 = 0.00; P > 0.001), mas em uma análise a nível de populações, a biomassa de rotíferos e cladóceros apresentou uma fraca correlação positiva. Copépodos Cyclopoida, os quais são considerados relativamente ineficientes na ingestão de cianobactérias, estiveram negativamente correlacionados com a concentração de microcistinas (r2 = - 0.01; P > 0.01). Surpreendentemente, a biomassa de copépodos Calanoida foi positivamente correlacionada com a concentração de cianotoxinas (r2 = 0.44; P > 0.001). Os resultados indicam que mecanismos de controle alelopáticos (efeitos negativos da microcistina sobre o zooplâncton) parecem não afetar substancialmente a composição do mesozooplâncton, que apresentou uma alta e constante biomassa, quando comparada à biomassa do microzooplâncton (rotíferos). Esses resultados podem ser importantes para um melhor entendimento das interações tróficas entre o zooplâncton e cianobactérias, e do efeito potencial de compostos alelopáticos sobre o zooplâncton.

Chemical modification of Aspergillus niger β-glucosidase and its catalytic properties

Aspergillus niger β-glucosidase was modified by covalent coupling to periodate activated polysaccharides (glycosylation). The conjugated enzyme to activated starch showed the highest specific activity (128.5 U/mg protein). Compared to the native enzyme, the conjugated form exhibited: a higher optimal reaction temperature, a lower Ea (activation energy), a higher Km (Michaelis constant) and Vmax (maximal reaction rate), and improved thermal stability. The calculated t1/2 (half-life) values of heat in-activation at 60 °C and 70 °C were 245.7 and 54.5 min respectively, whereas at these temperatures the native enzyme was less stable (t1/2 of 200.0 and 49.5 min respectively). The conjugated enzyme retained 32.3 and 29.7%, respectively from its initial activity in presence of 5 mM Sodium Dodecyl Sulphate (SDS) and p-Chloro Mercuri Benzoate (p-CMB), while the native enzyme showed a remarkable loss of activity (retained activity 1.61 and 13.7%, respectively). The present work has established the potential of glycosylation to enhance the catalytic properties of β-glucosidase enzyme, making this enzyme potentially feasible for biotechnological applications.