-
Proceedings of the National Academy of... May 2024Homomeric dimerization of metabotropic glutamate receptors (mGlus) is essential for the modulation of their functions and represents a promising avenue for the...
Homomeric dimerization of metabotropic glutamate receptors (mGlus) is essential for the modulation of their functions and represents a promising avenue for the development of novel therapeutic approaches to address central nervous system diseases. Yet, the scarcity of detailed molecular and energetic data on mGlu2 impedes our in-depth comprehension of their activation process. Here, we employ computational simulation methods to elucidate the activation process and key events associated with the mGlu2, including a detailed analysis of its conformational transitions, the binding of agonists, G protein coupling, and the guanosine diphosphate (GDP) release. Our results demonstrate that the activation of mGlu2 is a stepwise process and several energy barriers need to be overcome. Moreover, we also identify the rate-determining step of the mGlu2's transition from the agonist-bound state to its active state. From the perspective of free-energy analysis, we find that the conformational dynamics of mGlu2's subunit follow coupled rather than discrete, independent actions. Asymmetric dimerization is critical for receptor activation. Our calculation results are consistent with the observation of cross-linking and fluorescent-labeled blot experiments, thus illustrating the reliability of our calculations. Besides, we also identify potential key residues in the G protein binding position on mGlu2, mGlu2 dimer's TM6-TM6 interface, and Gi α5 helix by the change of energy barriers after mutation. The implications of our findings could lead to a more comprehensive grasp of class C G protein-coupled receptor activation.
Topics: Receptors, Metabotropic Glutamate; Humans; Protein Multimerization; Molecular Dynamics Simulation; Protein Conformation; Protein Binding
PubMed: 38739800
DOI: 10.1073/pnas.2401079121 -
JIMD Reports May 2024Three forms of muscular dystrophy-dystroglycanopathies are linked to the ribitol pathway. These include mutations in the isoprenoid synthase domain-containing protein...
Three forms of muscular dystrophy-dystroglycanopathies are linked to the ribitol pathway. These include mutations in the isoprenoid synthase domain-containing protein (), fukutin-related protein (), and fukutin () genes. The aforementioned enzymes are required for generation of the ribitol phosphate linkage in the O-glycan of alpha-dystroglycan. Mild cases of dystroglycanopathy present with slowly progressive muscle weakness, while in severe cases the eyes and brain are also involved. Previous research showed that ribose increased the intracellular concentrations of cytidine diphosphate-ribitol (CDP-ribitol) and had a therapeutic effect. Here, we report the safety and effects of oral ribose supplementation during 6 months in a patient with limb girdle muscular dystrophy type 2I (LGMD2I) due to a homozygous mutation. Ribose was well tolerated in doses of 9 g or 18 g/day. Supplementation with 18 g of ribose resulted in a decrease of creatine kinase levels of 70%. Moreover, metabolomics showed a significant increase in CDP-ribitol levels with 18 g of ribose supplementation ( < 0.001). Although objective improvement in clinical and patient-reported outcome measures was not observed, the patient reported subjective improvement of muscle strength, fatigue, and pain. This case study indicates that ribose supplementation in patients with dystroglycanopathy is safe and highlights the importance for future studies regarding its potential effects.
PubMed: 38736632
DOI: 10.1002/jmd2.12394 -
Se Pu = Chinese Journal of... Apr 2024Ibandronate sodium, a third-generation diphosphate drug used worldwide to treat osteoporosis, has the advantages of convenient use, low toxicity, and significant...
Ibandronate sodium, a third-generation diphosphate drug used worldwide to treat osteoporosis, has the advantages of convenient use, low toxicity, and significant therapeutic effects. However, the residual organic solvents in the synthesis process of sodium ibandronate not only have a negative impact on the efficacy of the drug, but also lead to a decrease in drug stability. Moreover, if the residual amounts of these solvents exceed safety standards, they may pose serious threats to human health. This study successfully established a convenient and efficient method based on headspace-gas chromatography (HS-GC) for the simultaneous determination of five residual solvents (methanol, acetone, benzene, toluene, 1-pentanol) in the raw materials of ibandronate sodium. The results indicated that satisfactory analytical performance can be achieved by using DB-624 capillary column (30 m×0.32 mm×1.8 μm) and a flame ionization detector in conjunction with headspace autosampling and a temperature program. The specific operating conditions included an initial temperature of 40 ℃, with a hold of 2 min, followed by a temperature ramp first to 200 ℃ at a rate of 5 ℃/min and then to 240 ℃ at a rate of 20 ℃/min, with a hold of 5 min. Nitrogen with a flow rate of 1 mL/min and split ratio of 14∶1 was used as the carrier gas. The headspace vial temperature was maintained at 80 ℃, and the sample equilibration time was 20 min. Under the established analytical conditions, good linear relationships were obtained between the mass concentrations of methanol (72-216 μg/mL), acetone (120-360 μg/mL), benzene (0.048-0.144 μg/mL), toluene (21.36-64.08 μg/mL), and 1-pentanol (120-360 μg/mL) and their corresponding peak areas, with correlation coefficients () greater than 0.990. The limits of detection for these solvents were 2.88, 0.011, 0.90, 0.24, and 0.024 ng/mL, respectively, with limits of quantification of 11.5, 0.043, 3.6, 0.96, and 0.096 ng/mL, respectively. Furthermore, the recoveries of these solvents ranged from 86.3% to 101.9%, with relative standard deviations (RSDs, =3) of less than 2.49%. The proposed method is simple, accurate, reliable, and suitable for the rapid and simultaneous determination of five residual solvents in the raw materials of ibandronate sodium. This study has important practical significance in improving drug safety and ensuring public health.
Topics: Chromatography, Gas; Solvents; Ibandronic Acid; Diphosphonates; Drug Contamination
PubMed: 38736392
DOI: 10.3724/SP.J.1123.2024.01023 -
Science Bulletin Apr 2024Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance....
Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance. TRPM2, a typical ancient ion channel, is activated by adenosine diphosphate ribose (ADPR) and calcium and its activation has evolved from a simple mode in invertebrates to a more complex one in vertebrates, but the evolutionary process is still unknown. Molecular evolutionary analysis of TRPM2s from more than 280 different animal species has revealed that, the C-terminal NUDT9-H domain has evolved from an enzyme to a ligand binding site for activation, while the N-terminal MHR domain maintains a conserved ligand binding site. Calcium gating pattern has also evolved, from one Ca-binding site as in sea anemones to three sites as in human. Importantly, we identified a new group represented by olTRPM2, which has a novel gating mode and fills the missing link of the channel gating evolution. We conclude that the TRPM2 ligand binding or activation mode evolved through at least three identifiable stages in the past billion years from simple to complicated and coordinated. Such findings benefit the evolutionary investigations of other channels and proteins.
PubMed: 38734586
DOI: 10.1016/j.scib.2024.04.052 -
Thrombosis Research Jun 2024Plaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII...
BACKGROUND
Plaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques.
AIMS
We aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits.
METHODS AND RESULTS
We assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5'-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group.
CONCLUSIONS
Increased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis.
Topics: Rabbits; Animals; Factor VIII; Neointima; Thrombosis; Male; Myocytes, Smooth Muscle; Tunica Intima; Humans; Platelet Aggregation; Femoral Artery
PubMed: 38729030
DOI: 10.1016/j.thromres.2024.04.025 -
Science Advances May 2024Cyanobacterial CO concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called...
Cyanobacterial CO concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium sp. PCC7001. Our results show that the CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.
Topics: Ribulose-Bisphosphate Carboxylase; Carbonic Anhydrases; Cyanobacteria; Allosteric Regulation; Phylogeny; Ribulosephosphates; Models, Molecular; Protein Multimerization; Carbon Dioxide; Substrate Specificity; Bacterial Proteins
PubMed: 38728392
DOI: 10.1126/sciadv.adk7283 -
Cells May 2024Ovarian cancer is a highly lethal form of gynecological cancer. This disease often goes undetected until advanced stages, resulting in high morbidity and mortality... (Review)
Review
Ovarian cancer is a highly lethal form of gynecological cancer. This disease often goes undetected until advanced stages, resulting in high morbidity and mortality rates. Unfortunately, many patients experience relapse and succumb to the disease due to the emergence of drug resistance that significantly limits the effectiveness of currently available oncological treatments. Here, we discuss the molecular mechanisms responsible for resistance to carboplatin, paclitaxel, polyadenosine diphosphate ribose polymerase inhibitors, and bevacizumab in ovarian cancer. We present a detailed analysis of the most extensively investigated resistance mechanisms, including drug inactivation, drug target alterations, enhanced drug efflux pumps, increased DNA damage repair capacity, and reduced drug absorption/accumulation. The in-depth understanding of the molecular mechanisms associated with drug resistance is crucial to unveil new biomarkers capable of predicting and monitoring the kinetics during disease progression and discovering new therapeutic targets.
Topics: Humans; Female; Ovarian Neoplasms; Drug Resistance, Neoplasm; Antineoplastic Agents
PubMed: 38727322
DOI: 10.3390/cells13090786 -
The Journal of Biological Chemistry Jun 2024In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is...
In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.
Topics: Escherichia coli; Escherichia coli Proteins; Fructokinases; Fructosediphosphates; Fructose; Gene Expression Regulation, Bacterial; Fructosephosphates
PubMed: 38723750
DOI: 10.1016/j.jbc.2024.107352 -
Microbiology Spectrum Jun 2024subsp. (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this...
UNLABELLED
subsp. (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the deletion mutant was unable to cause disease, whereas complementation restored wild-type phenotypes. XanB homology modeling allowed virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control.
IMPORTANCE
Xcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by sp.
Topics: Xanthomonas; Citrus; Plant Diseases; Bacterial Proteins; Nucleotidyltransferases; Biofilms; Virulence
PubMed: 38722158
DOI: 10.1128/spectrum.03673-23 -
Heliyon May 2024Antibiotic resistance and virulence profiles of , , and , isolated from water sources collected in informal settlements, were compared to clinical counterparts. Cluster...
Antibiotic resistance and virulence profiles of , , and , isolated from water sources collected in informal settlements, were compared to clinical counterparts. Cluster analysis using repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) indicated that, for each respective species, low genetic relatedness was observed between most of the clinical and environmental isolates, with only one clinical (PAO1) and one clinical (P2) exhibiting high genetic similarity to the environmental strains. Based on the antibiograms, the clinical Ef CD1 was extensively drug resistant (XDR); all isolates ( = 12) (except ATCC 13883) were multidrug resistant (MDR), while the ( = 16) isolates exhibited higher susceptibility profiles. The gene (tetracycline resistance) was identified in 47.4 % ( = 6 environmental; = 3 clinical) of the isolates, while the gene (carbapenem resistance) was detected in 52.6 % ( = 7 environmental; = 3 clinical) and 15.4 % ( = 2 environmental) of the and isolates, respectively. The isolates were predominantly poor biofilm formers, the isolates were moderate biofilm formers, while the isolates were strong biofilm formers. All and isolates were gamma (γ)-haemolytic, non-gelatinase producing ( only), and non-hypermucoviscous ( only), while the isolates exhibited beta (β)-haemolysis and produced gelatinase. The (type 1 fimbriae adhesion) and (uridine diphosphate galacturonate 4-epimerase synthesis) virulence genes were detected in the isolates, while the isolates possessed the (phenazine production) and (alginate biosynthesis) genes. Similarities in antibiotic resistance and virulence profiles of environmental and clinical , , and , thus highlights the potential health risks posed by using environmental water sources for daily water needs in low-and-middle-income countries.
PubMed: 38720709
DOI: 10.1016/j.heliyon.2024.e30215