Docking Site Research Articles

CDK activity at the centrosome regulates the cell cycle

In human cells and yeast, an intact "hydrophobic patch" substrate docking site is needed for mitotic cyclin centrosomal localization. A hydrophobic patch mutant (HPM) of the fission yeast mitotic cyclin Cdc13 cannot enter mitosis, but whether this is due to defective centrosomal localization or defective cyclin-substrate docking more widely is unknown. Here, we show that artificially restoring Cdc13-HPM centrosomal localization promotes mitotic entry and increases CDK (cyclin-dependent kinase) substrate phosphorylation at the centrosome and in the cytoplasm. We also show that the S-phase B-cyclin hydrophobic patch is required for centrosomal localization but not for S phase. We propose that the hydrophobic patch is essential for mitosis due to its requirement for the local concentration of cyclin-CDK with CDK substrates and regulators at the centrosome. Our findings emphasize the central importance of the centrosome as a hub coordinating cell-cycle control and explain why the cyclin hydrophobic patch is essential for mitosis.

MAPK-dependent control of mitotic progression in S. pombe

BackgroundMitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress.ResultsWe show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit.ConclusionsOur data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.

In Infected Nonunited Long-Bone Fractures of Lower Limbs, Bone Transport Through Induced Membrane Reduced Time to Docking Site Union and Risk of Nonunion and Infection Recurrence Versus Conventional Bone Transport.

In Infected Nonunited Long-Bone Fractures of Lower Limbs, Bone Transport Through Induced Membrane Reduced Time to Docking Site Union and Risk of Nonunion and Infection Recurrence Versus Conventional Bone Transport.

Refracture-related bone transport of tibia: technical notes and preliminary clinical results in nineteen cases.

Refracture is one of the main complications of bone transport, which brings additional physical and mental burden to surgeries and patients. We aimed to raise a new classification system of refracture-related bone transport based on the Simpson classification and to present our experience on treatment. This retrospective analysis included 19 patients with refracture-related bone transport (average age of 37.7years; 18 men). We developed a modified Simpson classification system to assist decision-making (conservative versus surgical). The ASAMI criteria were used to assess the outcomes at last follow-up. The mean follow-up was 12.3 ± 3.2months. Complete union was achieved in all patients, with no reinfection. Based on the modified Simpson classification, refracture was Ia type (within regeneration area) in three cases, Ib (collapsed fracture at the regeneration area) in one case, Ic (stress fracture) in three cases, II (at the junction between the regenerate and original bone) in one case, III (at the docking site) in nine cases, and V (at distant site) in two cases. Refracture was managed conservatively in six cases and surgically in 13 cases. Average time to bone union was 2.8 ± 1.2months in the conservative group versus 4.4 ± 1.4months in the surgery group. Assessment at the final follow-up using the ASAMI criteria revealed excellent bone result in all patients, excellent functional results in six patients (31.6%), and good functional results in 13 patients. The modified Simpson classification could include refracture at the docking site and stress fracture in the regeneration zone and provide some guidance in determining the appropriate treatment strategy.

Experimental Structures of Antibody/MHC-I Complexes Reveal Details of Epitopes Overlooked by Computational Prediction.

mAbs to MHC class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I Fabs bound to peptide/MHC-I/β2-microglobulin (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I α3 domain mAbs, and an anti-β2-microglobulin mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention.

Bioluminescence Resonance Energy Transfer (BRET)-based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly.

Two mother centrioles in an animal cell are linked by intercentriolar fibers that have CROCC/rootletin as their main building block. Here, we investigated the regulatory role of intercentriolar/rootlet fibers in cilia assembly. The cilia formation rates were significantly reduced in the CEP250/C-NAP1 and CROCC/rootletin knockout (KO) cells, irrespective of the departure of the young mother centrioles from the basal bodies. In addition, centriolar satellites were dispersed throughout the cytoplasm in the CEP250 and CROCC KO cells. We observed that PCM1 directly binds to CROCC. Their interaction is critical not only for the accumulation of centriolar satellites near the centrosomes/basal bodies but also for cilia formation. Finally, we observed that the centriolar satellite proteins are localized at the intercentriolar/rootlet fibers in the kidney epithelial cells. Based on these findings, we propose that the intercentriolar/rootlet fibers function as docking sites for centriolar satellites near the centrosomes/basal bodies and facilitate the cilia assembly process.

Induced membrane technique with plate fixation has a lower complication rate than bone transport over a plate for segmental tibial defects larger than 5cm.

The treatment of segmental tibial bone defects remains a surgical challenge. While Bone Transport (BT) and Induced Membrane Technique (IMT) are effective strategies for regenerating bone, there are few comparative studies between them. This investigation undertakes a comparative analysis of BT and IMT for large segmental tibial defects stabilised through plate fixation. Patients with segmental tibial defects exceeding 5cm were prospectively enrolled from 2008 to 2021 in a single institution, with a minimum follow-up duration of two years. All patients underwent either BT or IMT with plate fixation of the tibia. Procedural success, primary union as well as bone and functional outcome scores were compared. Complications, including non-unions, joint contractures and deep infections requiring surgical intervention, were also compared. 41 patients were recruited in total. 28 patients underwent Bone Transport Over a Plate (BTOP), while 13 patients underwent IMT with Plate fixation (IMTP). The procedural success rate trended higher in IMTP compared to BTOP (100% vs. 85.7%). The primary union rate also trended higher in IMTP compared to BTOP (92.3% vs. 79.2%). BTOP and IMTP achieved similar rates of satisfactory bone outcome scores (78.6% vs. 84.6%) and functional outcome scores (75% vs. 76.5%). There was no statistical difference between procedural success, primary union, bone and functional outcome scores. The complication rate in BTOP was 78.6% (22 of 28), including five docking site or regenerate non-unions, eight deep infections and nine joint contractures. IMTP had a 38.5% (5 of 13) complication rate, including one non-union, two deep infections and two joint contractures. The complication rate was 2.04 times higher in BTOP compared to IMTP (p = 0.0117). BTOP and IMTP are both equally effective techniques for regenerating bone in large tibial bone defects. However, IMTP may be a safer procedure than BTOP, with a lower probability of requiring additional procedures to address complications.

Treatment of Large Femoral and Tibial Bone Defects With Plate-Assisted Bone Segment Transport.

The purposes of this study were to assess clinical and radiographic outcomes following plate-assisted bone segment transport (PABST) in large bone defects of the lower extremities. Retrospective study of prospectively collected data. Level-1 trauma center located in Germany. Patients who underwent PABST and were at least 1 year postoperatively were included. Demographic data were collected. Radiographic apparent bone gap (RABG), time to consolidation, time to full weight-bearing, and consolidation index were calculated. Numeric rating scale, lower extremity functional scale (LEFS), and complications were assessed. Fifteen patients [13 male; mean age 51 years (range, 20-75)] underwent PABST and had follow-up at a mean of 29.1 months. The tibia was affected in 8 and the femur in 7 patients. Preoperative RABG was 60 mm [interquartile range (IQR): 40-125], and bone defects were caused by septic nonunions in 73% of patients. Fourteen patients (93%) demonstrated consolidated transport callus at 7.3 months [95% confidence interval (95% CI), 6-8.5], and 9 patients (60%) demonstrated complete consolidation of both docking site and transport callus at 11.5 months (95% CI, 7.3-15.3). Postoperative RABG was 0.1 mm (IQR: 0-0.8), and consolidation index was 1.9 months/cm (95% CI, 1.3-2.5). All patients achieved full weight-bearing at 8.7 months (IQR: 6.5-10.3). LEFS was 42 (95% CI, 34-50), and numeric rating scale was 3 (95% CI, 2-4). Patients treated for tibial defects had a significantly higher consolidation rate compared with patients treated for femoral defects ( P = 0.040). PABST demonstrated high consolidation of transport callus with few complications. Although full weight-bearing was achieved in all patients, complete consolidation of the docking site was only present in 60% of cases. Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Tepsin binds LC3B to promote ATG9A trafficking and delivery.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B preferentially over other members of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Reintroduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, reintroducing tepsin with a mutated LIR motif or missing N-terminus drives diffuse ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; ensuring delivery of ATG9A-positive vesicles; and in overall maintenance of autophagosome structure.

Nanoengineering Carboxysome Shells for Protein Cages with Programmable Cargo Targeting.

Protein nanocages have emerged as promising candidates for enzyme immobilization and cargo delivery in biotechnology and nanotechnology. Carboxysomes are natural proteinaceous organelles in cyanobacteria and proteobacteria and have exhibited great potential in creating versatile nanocages for a wide range of applications given their intrinsic characteristics of self-assembly, cargo encapsulation, permeability, and modularity. However, how to program intact carboxysome shells with specific docking sites for tunable and efficient cargo loading is a key question in the rational design and engineering of carboxysome-based nanostructures. Here, we generate a range of synthetically engineered nanocages with site-directed cargo loading based on an α-carboxysome shell in conjunction with SpyTag/SpyCatcher and Coiled-coil protein coupling systems. The systematic analysis demonstrates that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages could be precisely modulated by selecting specific anchoring systems and shell protein domains. Our study provides insights into the encapsulation principles of the α-carboxysome and establishes a solid foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability, thereby enabling applications in catalysis, delivery, and medicine.

Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

Network pharmacology and molecular dynamic simulation integrated strategy for the screening of active components and mechanisms of phytochemicals from Datura innoxia on Alzheimer and cognitive decline

Alzheimer’s disease (AD) ranks as the most prevalent neurodegenerative disorder with dementia and it accounts for more than 70% of all cases. Despite extensive reporting on the experimental investigation of Datura innoxia (DI) and its phytochemical components in the treatment of AD, the urgent need for elucidation of the principle of multi-mechanism and multi-level treatment of AD remains. In this research, molecular docking and network pharmacology were used to evaluate active compounds and molecular targets of DI for the treatment of AD. The phytochemical compounds of DI were obtained from the Indian Medicinal Plants, Phytochemistry, and Therapeutics (IMPPAT) as well as the Traditional Chinese Medicine System Pharmacology (TCMSP) databases. The screening includes the 28 most abundant components of DI and the Swiss Target Prediction database was used to predict targets of these compounds. The GeneCards database was used to collect AD-related genes. Both DI and AD targets were imported into a Venn diagram, and the 28 overlapped genes were identified as potential DI anti-AD targets. The results showed that Dinoxin B, Meteloidine, Scopoline, and Tropic acid had no effect on AD-related genes. Furthermore, the GO enrichment analysis indicates that DI influences molecular functions and biological processes such as learning or memory and modulation of chemical synaptic transmission as well as the membrane raft and membrane microdomain. The KEGG pathway analysis revealed that the key pathways implicated in DI's anti-AD actions include serotonergic synapse, IL-17 signaling pathway, and AGE-RAGE signaling pathway in diabetic complications. Based on the STRING and Cytoscape network-analysis platforms, the top ten anti-AD core targets include APP, CASP3, IL6, BACE1, IL1B, ACE, PSEN1, GAPDH, GSK3B and ACHE. The molecular docking and molecular dynamic simulation of the top two molecules against the top three target proteins confirmed the strong binding affinity and stability at the docked site. Overall, our findings pave the path for further research into the development and optimization of potential anti-AD agents from DI. Communicated by Ramaswamy H. Sarma

MLDSPP: Bacterial Promoter Prediction Tool Using DNA Structural Properties with Machine Learning and Explainable AI.

Bacterial promoters play a crucial role in gene expression by serving as docking sites for the transcription initiation machinery. However, accurately identifying promoter regions in bacterial genomes remains a challenge due to their diverse architecture and variations. In this study, we propose MLDSPP (Machine Learning and Duplex Stability based Promoter prediction in Prokaryotes), a machine learning-based promoter prediction tool, to comprehensively screen bacterial promoter regions in 12 diverse genomes. We leveraged biologically relevant and informative DNA structural properties, such as DNA duplex stability and base stacking, and state-of-the-art machine learning (ML) strategies to gain insights into promoter characteristics. We evaluated several machine learning models, including Support Vector Machines, Random Forests, and XGBoost, and assessed their performance using accuracy, precision, recall, specificity, F1 score, and MCC metrics. Our findings reveal that XGBoost outperformed other models and current state-of-the-art promoter prediction tools, namely Sigma70pred and iPromoter2L, achieving F1-scores >95% in most systems. Significantly, the use of one-hot encoding for representing nucleotide sequences complements these structural features, enhancing our XGBoost model's predictive capabilities. To address the challenge of model interpretability, we incorporated explainable AI techniques using Shapley values. This enhancement allows for a better understanding and interpretation of the predictions of our model. In conclusion, our study presents MLDSPP as a novel, generic tool for predicting promoter regions in bacteria, utilizing original downstream sequences as nonpromoter controls. This tool has the potential to significantly advance the field of bacterial genomics and contribute to our understanding of gene regulation in diverse bacterial systems.

Bioinformatics analysis and validation of the interaction between PML protein and TAB1 protein

To analyze the interaction between PML protein and TAB1 protein using bioinformatic approaches and experimentally verify the results. Using Rosetta software, a 3D model of TAB1 protein was constructed through a comparative modeling approach; the secondary structure of PML protein was retrieved in the PDB database and its crystal structure and 3D structure were resolved. Zdock 3.0.2 software was used to perform protein-protein docking of PML and TAB1, and the best conformation was extracted for molecular structure analysis of the docking model. The interaction between the two proteins was detected using immunoprecipitation in α-MMC-treated M1 inflammatory macrophages. When 6IMQ of PML was used as the docking site, PML protein formed 3 salt bridges, 6 hydrogen bonds and 6 hydrophobic interactions with TAB1 proteins; when 5YUF of PML was used as the docking site, PML protein formed 1 hydrogen bond, 3 electrostatic interactions and 9 hydrophobic interactions with TAB1 proteins, and both of the docking modes formed good molecular docking and interactions. In the M1 inflammatory macrophages treated with α-MMC for 4 h, positive protein bands of PML and TAB1 were detected in the cell lysates in PML-IP group. PML protein can interact strongly with TAB1 protein.

Histone globular domain epigenetic modifications: The regulators of chromatin dynamics in malaria parasite.

Plasmodium species adapt a complex lifecycle with multiple phenotypes to survive inside various cell types of humans and mosquitoes. Stage-specific gene expression in the developmental stages of parasites is tightly controlled in Plasmodium species; however, the underlying mechanisms have yet to be explored. Genome organization and gene expression for each stage of the malaria parasite need to be better characterized. Recent studies indicated that epigenetic modifications of histone proteins play a vital role in chromatin plasticity. Like other eukaryotes, Plasmodium species N-terminal tail modifications form a distinct "histone code," which creates the docking sites for histone reader proteins, including gene activator/repressor complexes, to regulate gene expression. The emerging research findings shed light on various unconventional epigenetic changes in histone proteins' core/globular domain regions, which might contribute to the chromatin organization in different developmental stages of the malaria parasite. The malaria parasite lost many transcription factors during evolution, and it is proposed that the nature of local chromatin structure essentially regulates the stage-specific gene expression. This review highlights recent discoveries of unconventional histone globular domain epigenetic modifications and their functions in regulating chromatin structure dynamics in various developmental stages of malaria parasites.

Bone transport combined with sequential nailing technique for the management of large segmental bone defects after trauma.

Bone transport technique is widely used for the management of large segmental bone defects. However, several reasons may prevent its successful completion, such as poor osteogenesis, docking site nonunion, severe chronic pain and psychological problems. We used sequential nailing technique to solve these problems. The objective of this study was to analyze the clinical effects of our modified technique for the management of large segmental bone defects after trauma. Twenty-three patients using bone transport combined with sequential nailing technique in our institution from June 2011 to June 2020 were included and analyzed retrospectively. There were 15 males and eight females. The age ranged from 19 to 64 years. There were eight cases suffering from basic medical diseases. The initial injury was open in 14 patients. Seven cases encountered femoral defects and 16 for tibia. The main reasons for sequential nailing technique were docking site nonunion (nine cases), poor osteogenesis (five cases), severe chronic pain (five cases) and psychological problems (four cases). The residual bone defects after removing the external fixator, operation plans, complications and follow-up time were recorded. Bone defect healing was evaluated by Paley score. The mean residual bone defects were (2.9 ± 1.9) cm. The mean time in external fixator was (9.5 ± 3.4) months. The average follow-up time was (23 ± 3) months. With respect to complications, two cases suffered from nonunion again and were treated by bone graft with augmented plate fixation. No infection recurrence was found in these cases. The excellent and good rate of bone defect healing was 91.3%. Bone transport combined with sequential nailing technique could shorten the external fixation time, overcome the inconvenience of the external frame to patients, eliminate chronic pain and be easy for patients to accept. Patients using this modified technique achieved high satisfaction.

A maximum of two readily releasable vesicles per docking site at a cerebellar single active zone synapse.

Recent research suggests that in central mammalian synapses, active zones contain several docking sites acting in parallel. Before release, one or several synaptic vesicles (SVs) are thought to bind to each docking site, forming the readily releasable pool (RRP). Determining the RRP size per docking site has important implications for short-term synaptic plasticity. Here, using mouse cerebellar slices, we take advantage of recently developed methods to count the number of released SVs at single glutamatergic synapses in response to trains of action potentials (APs). In each recording, the number of docking sites was determined by fitting with a binomial model the number of released SVs in response to individual APs. After normalization with respect to the number of docking sites, the summed number of released SVs following a train of APs was used to estimate of the RRP size per docking site. To improve this estimate, various steps were taken to maximize the release probability of docked SVs, the occupancy of docking sites, as well as the extent of synaptic depression. Under these conditions, the RRP size reached a maximum value close to two SVs per docking site. The results indicate that each docking site contains two distinct SV-binding sites that can simultaneously accommodate up to one SV each. They further suggest that under special experimental conditions, as both sites are close to full occupancy, a maximal RRP size of two SVs per docking site can be reached. More generally, the results validate a sequential two-step docking model previously proposed at this preparation.

A maximum of two readily releasable vesicles per docking site at a cerebellar single active zone synapse

Recent research suggests that in central mammalian synapses, active zones contain several docking sites acting in parallel. Before release, one or several synaptic vesicles (SVs) are thought to bind to each docking site, forming the readily releasable pool (RRP). Determining the RRP size per docking site has important implications for short-term synaptic plasticity. Here, using mouse cerebellar slices, we take advantage of recently developed methods to count the number of released SVs at single glutamatergic synapses in response to trains of action potentials (APs). In each recording, the number of docking sites was determined by fitting with a binomial model the number of released SVs in response to individual APs. After normalization with respect to the number of docking sites, the summed number of released SVs following a train of APs was used to estimate of the RRP size per docking site. To improve this estimate, various steps were taken to maximize the release probability of docked SVs, the occupancy of docking sites, as well as the extent of synaptic depression. Under these conditions, the RRP size reached a maximum value close to two SVs per docking site. The results indicate that each docking site contains two distinct SV-binding sites that can simultaneously accommodate up to one SV each. They further suggest that under special experimental conditions, as both sites are close to full occupancy, a maximal RRP size of two SVs per docking site can be reached. More generally, the results validate a sequential two-step docking model previously proposed at this preparation.

Diphenolic boldine, an aporphine alkaloid: inhibitory effect evaluation on α-glucosidase by molecular dynamics integrating enzyme kinetics

Screening α-glucosidase inhibitors with novel structures is an important field in the development of anti-diabetic drugs due to their application in postprandial hyperglycemia control. Boldine is one of the potent natural antioxidants with a wide range of pharmacological activities. Virtual screening and biochemical inhibition kinetics combined with molecular dynamics simulations were conducted to verify the inactivation function of boldine on α-glucosidase. A series of inhibition kinetics and spectrometry detections were conducted to analyze the α-glucosidase inhibition. Computational simulations of molecular dynamics/docking analyses were conducted to detect boldine docking sites’ details and evaluate the key binding residues. Boldine displayed a typical reversible and mixed-type inhibition manner. Measurements of circular dichroism and fluorescence spectrum showed boldine changed the secondary structure and loosened the tertiary conformation of target α-glucosidase. The computational molecular dynamics showed that boldine could block the active pocket site through close interaction with binding key residues, and two phenolic hydroxyl groups of boldine play a core function in α-glucosidase inhibition via ligand binding. This investigation reveals the boldine function on interaction with the α-glucosidase active site, which provides a new inhibitor candidate. Communicated by Ramaswamy H. Sarma