Positive Cooperativity Research Articles

The Kinetics of Carbon-Carbon-Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity.

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b-ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon-carbon-bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS-mediated carbon-carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate-limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS-mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

The Kinetics of Carbon‐Carbon‐Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b‐ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon‐carbon‐bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS‐mediated carbon‐carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate‐limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS‐mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

Allostery in homodimeric SARS-CoV-2 main protease

Many enzymes work as homodimers with two distant catalytic sites, but the reason for this choice is often not clear. For the main protease Mpro of SARS-CoV-2, dimerization is essential for function and plays a regulatory role during the coronaviral replication process. Here, to analyze a possible allosteric mechanism, we use X-ray crystallography, native mass spectrometry, isothermal titration calorimetry, and activity assays to study the interaction of Mpro with three peptide substrates. Crystal structures show how the plasticity of Mpro is exploited to face differences in the sequences of the natural substrates. Importantly, unlike in the free form, the Mpro dimer in complex with these peptides is asymmetric and the structures of the substrates nsp5/6 and nsp14/15 bound to a single subunit show allosteric communications between active sites. We identified arginines 4 and 298 as key elements in the transition from symmetric to asymmetric dimers. Kinetic data allowed the identification of positive cooperativity based on the increase in the processing efficiency (kinetic allostery) and not on the better binding of the substrates (thermodynamic allostery). At the physiological level, this allosteric behavior may be justified by the need to regulate the processing of viral polyproteins in time and space.

Lapachol, a natural food component, interacts with human serum albumin: Insights of its impact on the pharmacokinetics of clinically used drugs

Lapachol (LAP), a natural 1,4-naphthoquinone used in popular medicine of South American, is an antioxidant and antimicrobial compound used as a food additive; however, its interactive profile with the main protein carrier of compounds in the human bloodstream (human serum albumin, HSA) was not still characterized. Additionally, the impact of LAP in binding clinically drugs to albumin is still unknown. Thus, the present work describes the interaction HSA:LAP using different biophysical techniques, i.e., 1H saturation-transfer difference nuclear magnetic resonance (1H STD-NMR), isothermal titration calorimetry (ITC), steady-state and time-resolved fluorescence measurements combined with molecular docking calculations. LAP interacts with subdomain region IIA (site I), mainly driven by enthalpy effects, while subdomain region IB (site III) was identified as the second binding site, mainly driven by entropy effects. The binding is spontaneous, strong (binding constant average, Kaverage ≈ 4.45 × 105 M−1), and there is a positive cooperativity in the presence of ibuprofen, with the LAP structure fully buried into the protein cavities. Overall, LAP might impact the residence time (pharmacokinetic profile) of drugs that bind to subdomains regions IIA and IB of albumin, e.g., warfarin, phenylbutazone, diflunisal, naproxen, camptothecin, doxorubicin, daunorubicin, suramin, and tyrosine kinase inhibitors.

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest (∼10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both α-helix and β-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a Gx3Sx4G motif homologous to canonical ‘GxSxG’ motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

DEA와 SFA 모형을 이용한 한국 프로야구의 비용효율성 분석

The purpose of this study is to analyze the cost efficiency of Korean professional baseball(KBO) teams through Data Envelopment Analysis(DEA) and Stochastic Frontier Analysis(SFA) and to examine its determinants.. In this study, we used 20 years of data from 2003 to 2022 for eight teams to examine the cost efficiency and its influencing factors of each team through DEA window analysis and SFA reflecting the time series. The empirical results showed that the cost efficiency of KBO teams is not high, but it is generally improving. Among them, the size of the stadium and the number of wins have a positive effect on cost efficiency, while the number of new stadiums and free agents has a negative effect on cost efficiency. In particular, the difference between the efficiency values derived from the two methods is quite large, which means that it is highly sensitive to external shocks due to the probable error. The fact that many teams react greatly to external shocks suggests that the support of parent companies and local governments greatly affects the sustainability of the teams due to the lack of self-reliance of the teams. In order to increase the self-reliance of KBO teams facing a new competitive environment after COVID-19, continuous efforts are needed to transform into solid companies, including positive external support and cooperation with local governments, as well as creating higher-quality cultural content and discovering diverse sources of revenue.

Vitis vinifera Lipoxygenase LoxA is an Allosteric Dimer Activated by Lipidic Surfaces

Lipoxygenases catalyze the peroxidation of poly-unsaturated fatty acid chains either free or esterified in membrane lipids. Vitis vinifera LoxA is transcriptionally induced at ripening onset and localizes at the inner chloroplastic membrane where it is responsible for galactolipid regiospecific mono- and di-peroxidation. Here we present a kinetic and structural characterization of LoxA. Our X-ray structures reveal a constitutive dimer with detergent induced conformational changes affecting substrate binding and catalysis. In a closed conformation, a LID domain prevents substrate access to the catalytic site by steric hindrance. Detergent addition above the CMC destabilizes the LID and opens the dimer with both catalytic sites accessible from the same surface framed by the PLAT domains. As a consequence, detergent molecules occupy allosteric sites in the PLAT/catalytic domain interfaces. These structural changes are mirrored by increased enzymatic activity and positive cooperativity when the substrate is provided in micelles. The ability to interact with micelles is lost upon dimer destabilization by site-directed mutagenesis as assessed by tryptophan fluorescence. Our data allow to propose a model for protein activation at the membrane, classifying LoxA as an interfacial enzyme acting on fatty acid chains directly from the membrane similar to mammalian 15-LOX and 5-LOX.

Connectedness of Malaysian Students in Indonesia Through Social Media

This research identifies the motivation behind the connection between Malaysian students studying at the Faculty of Da'wah and Communication at UIN Sunan Kalijaga Yogyakarta through social media. This research aims to provide recommendations to strengthen positive cooperation in the field of education between Malaysia and Indonesia. Although much research has been conducted on the Malaysian diaspora, this paper is novel by highlighting a specific aspect of the subject that rarely receives attention, namely the diaspora who are still students. Using a case study approach, the qualitative research method uses interviews as a data collection tool based on the Uses and Gratification theory. The research results show that Malaysian students are connected through social media with several motivations, such as diversion, personal relationships, personal identity, and surveillance. They use social media as an escape through culinary content, a means of sharing recommendations for tourist attractions, strengthening religious and national identity, and monitoring current conditions in both Indonesia and Malaysia.

Rationalize the Functional Roles of Protein-Protein Interactions in Targeted Protein Degradation by Kinetic Monte-Carlo Simulations.

Targeted protein degradation is a promising therapeutic strategy to tackle disease-causing proteins that lack binding pockets for traditional small-molecule inhibitors. Its first step is to trigger the proximity between a ubiquitin ligase complex and a target protein through a heterobifunctional molecule, such as proteolysis targeting chimeras (PROTACs), leading to the formation of a ternary complex. The properties of protein-protein interactions play an important regulatory role during this process, which can be reflected by binding cooperativity. Unfortunately, although computer-aided drug design has become a cornerstone of modern drug development, the endeavor to model targeted protein degradation is still in its infancy. The development of computational tools to understand the impacts of protein-protein interactions on targeted protein degradation, therefore, is highly demanded. To reach this goal, we constructed a non-redundant structural benchmark of the most updated ternary complexes and applied a kinetic Monte-Carlo method to simulate the association between ligases and PROTAC-targeted proteins in the benchmark. Our results show that proteins in most complexes with positive cooperativity tend to associate into native-like configurations more often. In contrast, proteins very likely failed to associate into native-like configurations in complexes with negative cooperativity. Moreover, we compared the protein-protein association through different interfaces generated from molecular docking. The native-like binding interface shows a higher association probability than all the other alternative interfaces only in the complex with positive cooperativity. These observations support the idea that the formation of ternary complexes is closely regulated by the binary interactions between proteins. Finally, we applied our method to cyclin-dependent kinases 4 and 6 (CDK4/6). We found that their interactions with the ligase are not as similar as their structures. Altogether, our study paves the way for understanding the role of protein-protein interactions in PROTACE-induced ternary complex formation. It can potentially help in searching for degraders that selectively target specific proteins.

PENERAPAN PEMBELAJARAN PROBLEM BASED LEARNING MELALUI GAME PUZZLE DALAM MENINGKATKAN NILAI KERJASAMA PADA MATA PELAJARAN PENDIDIKAN PANCASILA

The background of this study is the low attitude of students working class X-TKRO 2 SMKN 3 Surabaya. This study aims to increase the value of positive cooperation for learners with problem based learning (PBL) through puzzle games on Pancasila education subjects. The type of research used is class action research with two cycles. The subjects of this study were students of Class X-TKRO 2. Data analysis techniques performed using descriptive analysis techniques. Hasi this study addresses the value of cooperation of students in Cycle 1 is 67,62% and Cycle 2 have increased 83,38%. The results of this study show that the application of problem based learning (PBL) through puzzle games can increase the value of cooperation of students in Pancasila education subjects.

Atomic-Level Free Energy Landscape Reveals Cooperative Symport Mechanism of Melibiose Transporter.

The Major Facilitator Superfamily (MFS) transporters are an essential class of secondary active transporters involved in various physiological and pathological processes. The melibiose permease (MelB), which catalyzes the stoichiometric symport of the disaccharide melibiose and monovalent cations (e.g., Na+, H+, or Li+), is a key model for understanding the cation-coupled symport mechanisms. Extensive experimental data has established that positive cooperativity between the cargo melibiose and the coupling cation is central to the symport mechanism. However, the structural and energetic origins of this cooperativity remain unclear at the atomistic level for MelB and most other coupled transporters. Here, leveraging recently resolved structures in inward- and outward-facing conformations, we employed the string method and replica-exchange umbrella sampling simulation techniques to comprehensively map the all-atom free energy landscapes of the Na+-coupled melibiose translocation across the MelB in Salmonella enterica serovar Typhimurium (MelBSt), in comparison with the facilitated melibiose transport in a uniporter mutant. The simulation results unravel asymmetrical free energy profiles of melibiose translocation, which is tightly coupled to protein conformational changes in both the N- and C-terminal domains. Notably, the cytoplasmic release of the melibiose induces the simultaneous opening of an inner gate, resulting in a high-energy state of the system. Periplasmic sugar binding and cytoplasmic melibiose released are dynamically coupled with changes in the internal gating elements along the translocation pathway. The outward-facing sugar-bound state is thermodynamically most stable, while the occluded state is a transient state. The binding of Na+ facilitates melibiose translocation by increasing the melibiose-binding affinity and decreasing the overall free energy barrier and change. The cooperative binding of the two substrates results from the allosteric coupling between their binding sites instead of direct electrostatic interaction. These findings add substantial new atomic-level details into how Na+ binding facilitates melibiose translocation and deepen the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters.

The Legal Dilemma and Optimization Path of the Jurisdiction of the International Court of Justice in Litigation

The litigation of the International Court of Justice (ICJ) is a vital avenue for peacefully resolving legal disputes between nations and fostering positive international cooperation. ICJs aim is to refine and develop its methodology for properly handling and adjudicating various legal disputes among countries, and to optimize the related codified rule framework accordingly. Concurrently, the ICJ harbors the vision of broadening its jurisdiction and gaining recognition from more countries, striving to better maintain international peace, promote the proper resolution of international disputes, and advance the continuous progress and development of the international judicial system. However, in the context of the evolving and progressing international community, the ICJ has encountered numerous challenges in the actual exercise of its jurisdictional authority. This study examines these challenges in depth, focusing on issues related to admissibility, optional compulsory jurisdiction, and delayed jurisdiction. Through this analysis, a series of targeted recommendations are proposed to address these practical dilemmas and to support the ICJ in fulfilling its crucial mandate more effectively.

학술지 『제주도연구』(1984~2023)의 연구동향 분석 - 제주교육학의 관점에서

The purpose of this study was to analyze specifically the research trends of ‘Journal of Jeju Studies [JJS]’. To accomplish it, the study selected 515 articles from JJS, that had been published from 1981 to 2023, and analyzed them by Keyword Network Analysis. Major results of the study are as follows: First, it needs to cultivate more positive communications and cooperation amongst humanities, social science and natural science in order to vitalize interdisciplinary studies of the three academic areas, that is a major strength of JJS. Second, the various opportunities should be provided positively for educational researchers to pursue the integral studies by harmonizing the uniqueness and generalities of Jeju education. Third, although research subjects of JJS are different according to the time period, the major keywords such as culture, region, society, and tourism were ranked highly by the analysis results. Therefore, it is necessary to support the researchers and to cultivate research environment in order to expand the spectrum of research subjects. Lastly, the future studies of Jeju education need to connect to social science and to link to humanities beyond educational study realm itself. The results would be used as the basic implications for the further researches of Jeju as well Jeju education in terms of research subjects and research directions.

Regioselectivity and physical nature of the interactions between (methyl)guanine with HCl and CH3OH

A comprehensive study of the hydrogen bonding interactions between guanine (G) and methyl guanine derivatives (MGs) in the presence of HCl and MeOH is carried out at B3LYP, B3LYP-D3 and M062X/6–311 + + G(d.p) levels using molecular electrostatic potential, natural bond orbital, and symmetry adapted perturbation theory. Making use of these state-of-the-art techniques, this study attempts to elucidate the chemical bonding, regioselectivity, and physical nature of the interactions responsible for the stability of (M)G…L (L = HCl, MeOH) acid–base complexes. Our calculations reveal that 1-G, 3-MG, and 5-MG interact more strongly with MeOH than HCl due to the positive hydrogen bond cooperativity. Furthermore, the carbonyl site on G is found to be the most reactive site, while methyl substitution increases the basicity of the nucleobase, thus yieding more stable complexes. The strongest H-bond interaction in G-complexes is found when HCl and MeOH attack the carbonyl group in anti-position. Finally, energy decomposition analyses through the symmetry-adapted perturbation theory reveal that most complexes are mainly stabilized via electrostatic interactions. The energy difference between complex isomers shows a competition between 3-HCl-G (MG) and 4-HCl-G (MG) at ∆G level where thermal, BSSE and entropy terms are included.

Positive cooperativity in synergistic activation of Wnt proteins

BackgroundWnt proteins are crucial for embryonic development, stem cell growth, and tissue regeneration. Wnt signaling pathway is activated when Wnt proteins bind to cell membrane receptors.Methods and resultsWe employed a luciferase reporter assay in HEK293STF cells to measure Wnt protein-induced signaling. We observed that Wnt3a uniquely promotes the Wnt/β-catenin pathway through positive cooperativity. Additionally, MFH-ND, a molecular mimic of Wnt ligands, markedly increased Wnt3a-induced signaling in a dose-responsive manner. This suggests that various Wnt ligands can synergistically enhance Wnt pathway activation.ConclusionsThe study suggests the likelihood of various Wnt ligands coexisting in a single signalosome on the cell membrane, providing new insights into the complexities of Wnt signaling mechanisms.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

Stimuli‐Responsive Molecular Duplexes Displaying Duplex‐to‐Duplex Switching

AbstractSynthetic duplexes with high stabilities have promising potential for mimicking biomolecular functions and developing supramolecular smart materials. Herein, we describe the synthesis and stimuli‐responsive properties of molecular duplexes derived from indolocarbazole–pyridine (I−P) oligomers. These duplexes adopt nonclassical helical structures, stabilized by I−P hydrogen‐bonding pairs in anhydrous chlorinated solvents. Notably, the longest duplex 62 (11‐mer)2 displays remarkable stability, forming twenty hydrogen bonds; its exchange energy barrier was determined to be ΔG≠=22.0 kcal ⋅ mol−1 at 75 °C in anhydrous (CDCl2)2. Upon the addition of water, a hydrated duplex 62 (11‐mer)2⊃10H2O was formed, with one water molecule inserted between each I−P hydrogen‐bonding pair. The Hill coefficient (n) for this process is 6.1, demonstrating extremely positive cooperativity. Conversely, the hydrated duplex 62 (11‐mer)2⊃10H2O was completely converted into the original anhydrous duplex 62 (11‐mer)2 when the temperature was increased. Interconversion between these two distinct duplexes can be repeatedly carried out by varying the temperature. Furthermore, reversible switching between hetero‐duplexes and homo‐duplexes was also demonstrated by controlling the temperature, with concomitant changes in the characteristic emission signals.

Stimuli-Responsive Molecular Duplexes Displaying Duplex-to-Duplex Switching.

Synthetic duplexes with high stabilities have promising potential for mimicking biomolecular functions and developing supramolecular smart materials. Herein, we describe the synthesis and stimuli-responsive properties of molecular duplexes derived from indolocarbazole-pyridine (I-P) oligomers. These duplexes adopt nonclassical helical structures, stabilized by I-P hydrogen-bonding pairs in anhydrous chlorinated solvents. Notably, the longest duplex 62 (11-mer)2 displays remarkable stability, forming twenty hydrogen bonds; its exchange energy barrier was determined to be ΔG≠=22.0 kcal ⋅ mol-1 at 75 °C in anhydrous (CDCl2)2. Upon the addition of water, a hydrated duplex 62 (11-mer)2⊃10H2O was formed, with one water molecule inserted between each I-P hydrogen-bonding pair. The Hill coefficient (n) for this process is 6.1, demonstrating extremely positive cooperativity. Conversely, the hydrated duplex 62 (11-mer)2⊃10H2O was completely converted into the original anhydrous duplex 62 (11-mer)2 when the temperature was increased. Interconversion between these two distinct duplexes can be repeatedly carried out by varying the temperature. Furthermore, reversible switching between hetero-duplexes and homo-duplexes was also demonstrated by controlling the temperature, with concomitant changes in the characteristic emission signals.

Phosphate-dependent nuclear export via a novel NES class recognized by exportin Msn5.

Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Unlike the traditional hydrophobic nuclear export signal (NES) utilized by the Exportin-1/XPO1 system, cryogenic-electron microscopy structures reveal that Pho4 presents a novel, phosphorylated 35-residue NES that interacts with the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches, unveiling a previously unknown mechanism of phosphate-specific recognition. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4's release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.

Retinopathy of Prematurity and Bioinformatics Analysis: Bibliometric Studies and Visual Analysis by Cite Space

Objective: The study aims to explore the hotspots and frontiers of retinopathy of prematurity (ROP) and bioinformatics analysis by reviewing the current status by Cite Space. Methods: The Web of Science database (WoS) was searched from ROP to 2010. Cite Space is used to generate network maps about the collaboration between authors, countries and institutions and to reveal hotspots and frontiers of both. Results: 98 studies related to ROP and various bioinformatics analyses were retrieved from WoS. North-Eastern University and Massachusetts General Hospital are the major countries and institutions. Hot topics focus on the interaction between the two, and possible new diagnostic and control measures. Conclusion: based on the results of the Cite Space study, scholars suggest that the deepening of the authors, positive cooperation between countries and institutions, mainly committed to research including artificial intelligence deep learning and the mutual recognition of biological information, especially through vascularization, additional lesions, classification and gene expression, this may mean that the future may be rapid and accurate diagnosis of ROP, especially invasive retinopathy of premature maturity (A-ROP).