A ferritin nanoparticle vaccine based on the hemagglutinin extracellular domain of swine influenza A (H1N1) virus elicits protective immune responses in mice and pigs.

Introduction: Swine influenza viruses (SIVs) pose significant economic losses to the pig industry and are a burden on global public health systems. The increasing complexity of the distribution and evolution of different serotypes of influenza strains in swine herds escalates the potential for the emergence of novel pandemic viruses, so it is essential to develop new vaccines based on swine influenza. Methods: Here, we constructed a self-assembling ferritin nanoparticle vaccine based on the hemagglutinin (HA) extracellular domain of swine influenza A (H1N1) virus using insect baculovirus expression vector system (IBEVS), and after two immunizations, the immunogenicities and protective efficacies of the HA-Ferritin nanoparticle vaccine against the swine influenza virus H1N1 strain in mice and piglets were evaluated. Results: Our results demonstrated that HA-Ferritin nanoparticle vaccine induced more efficient immunity than traditional swine influenza vaccines. Vaccination with the HA-Ferritin nanoparticle vaccine elicited robust hemagglutinin inhibition titers and antigen-specific IgG antibodies and increased cytokine levels in serum. MF59 adjuvant can significantly promote the humoral immunity of HA-Ferritin nanoparticle vaccine. Furthermore, challenge tests showed that HA-Ferritin nanoparticle vaccine conferred full protection against lethal challenge with H1N1 virus and significantly decreased the severity of virus-associated lung lesions after challenge in both BALB/c mice and piglets. Conclusion: Taken together, these results indicate that the hemagglutinin extracellular-based ferritin nanoparticle vaccine may be a promising vaccine candidate against SIVs infection.

Optimizing recombinant antibody fragment production: A comparison of artificial intelligence and statistical modeling.

Maximizing the recombinant protein yield necessitates optimizing the production medium. This can be done using a variety of methods, including the conventional "one-factor-at-a-time" approach and more recent statistical and mathematical methods such as artificial neural network (ANN), genetic algorithm, etc. Every approach has advantages and disadvantages of its own, yet even when a technique has flaws, it is nevertheless used to get the best results. Here, one categorical variable and four numerical parameters, including post-induction time, inducer concentration, post-induction temperature, and pre-induction cell density, were optimized using the 232 experimental assays of the central composite design. The direct and indirect effects of factors on the yield of anti-epithelial cell adhesion molecule extracellular domain fragment antibody were examined using statistical methods. The analysis of variance results indicate that the response surface methodology (RSM) model is effective in predicting the amount of produced single-chain fragment variable (p-value = 0.0001 and R2 = 0.905). For ANN modeling, the evaluation using normalized root mean square error (NRMSE) and R2 values shows a good fit (R2 = 0.942) and accurate predictions (NRMSE = 0.145). The analysis of error parameters and R2 of a dataset, which contained 30 data points randomly selected from the complete dataset, showed that the ANN model had a higher R2 value (0.968) compared to the RSM model (0.932). Furthermore, the ANN model demonstrated stronger predictive ability with a lower NRMSE (0.048 vs. 0.064). Induction at the cell density of 0.7 and an isopropyl ß-D-1-thiogalactopyranoside concentration of 0.6 mM for 32 h at 30°C in BW25113 was the ideal culture condition leading to the protein yield of 259.51 mg/L. Under the optimum conditions, the output values predicted by the ANN model (259.83 mg/L) were more in line with the experimental data (259.51 mg/L) than the RSM (276.13 mg/L) expected value. This outcome demonstrated that the ANN model outperforms the RSM in terms of prediction accuracy.

Insight into the autoproteolysis mechanism of the RsgI9 anti-σ factor from Clostridium thermocellum.

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/ß/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

Unraveling the Interplay of Extracellular Domain Conformational Changes and Parathyroid Hormone Type 1 Receptor Activation in Class B1 G Protein-Coupled Receptors: Integrating Enhanced Sampling Molecular Dynamics Simulations and Markov State Models.

Parathyroid hormone (PTH) type 1 receptor (PTH1R), as a typical class B1 G protein-coupled receptor (GPCR), is responsible for regulating bone turnover and maintaining calcium homeostasis, and its dysregulation has been implicated in the development of several diseases. The extracellular domain (ECD) of PTH1R is crucial for the recognition and binding of ligands, and the receptor may exhibit an autoinhibited state with the closure of the ECD in the absence of ligands. However, the correlation between ECD conformations and PTH1R activation remains unclear. Thus, this study combines enhanced sampling molecular dynamics (MD) simulations and Markov state models (MSMs) to reveal the possible relevance between the ECD conformations and the activation of PTH1R. First, 22 intermediate structures are generated from the autoinhibited state to the active state and conducted for 10 independent 200 ns simulations each. Then, the MSM is constructed based on the cumulative 44 µs simulations with six identified microstates. Finally, the potential interplay between ECD conformational changes and PTH1R activation as well as cryptic allosteric pockets in the intermediate states during receptor activation is revealed. Overall, our findings reveal that the activation of PTH1R has a specific correlation with ECD conformational changes and provide essential insights for GPCR biology and developing novel allosteric modulators targeting cryptic sites.

Progress to Clarify How NOTCH3 Mutations Lead to CADASIL, a Hereditary Cerebral Small Vessel Disease.

Notch signaling is conserved in C. elegans, Drosophila, and mammals. Among the four NOTCH genes in humans, NOTCH1, NOTCH2, and NOTCH3 are known to cause monogenic hereditary disorders. Most NOTCH-related disorders are congenital and caused by a gain or loss of Notch signaling activity. In contrast, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 is adult-onset and considered to be caused by accumulation of the mutant NOTCH3 extracellular domain (N3ECD) and, possibly, by an impairment in Notch signaling. Pathophysiological processes following mutant N3ECD accumulation have been intensively investigated; however, the process leading to N3ECD accumulation and its association with canonical NOTCH3 signaling remain unknown. We reviewed the progress in clarifying the pathophysiological process involving mutant NOTCH3.

Analysis of Receptor-Type Protein Tyrosine Phosphatase Extracellular Regions with Insights from AlphaFold.

The receptor-type protein tyrosine phosphatases (RPTPs) are involved in a wide variety of physiological functions which are mediated via their diverse extracellular regions. They play key roles in cell-cell contacts, bind various ligands and are regulated by dimerization and other processes. Depending on the subgroup, they have been described as everything from 'rigid rods' to 'floppy tentacles'. Here, we review current experimental structural knowledge on the extracellular region of RPTPs and draw on AlphaFold structural predictions to provide further insights into structure and function of these cellular signalling molecules, which are often mutated in disease and are recognised as drug targets. In agreement with experimental data, AlphaFold predicted structures for extracellular regions of R1, and R2B subgroup RPTPs have an extended conformation, whereas R2B RPTPs are twisted, reflecting their high flexibility. For the R3 PTPs, AlphaFold predicts that members of this subgroup adopt an extended conformation while others are twisted, and that certain members, such as CD148, have one or more large, disordered loop regions in place of fibronectin type 3 domains suggested by sequence analysis.

Dimerization of the CNNM extracellular domain.

Cystathionine- ß $$ \beta $$ -synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They mediate magnesium homeostasis directly by transport of Mg2+ ions and indirectly by regulation of the transient receptor potential ion channel subfamily M member 7 (TRPM7). Here, we report the crystal structure of the extracellular domain of tapeworm CNNM4. The domain forms a dimer of immunoglobulin-like (Ig-like) folds with electron density observed for three glycosylation sites. Analytical ultracentrifugation confirms that mutations in the extracellular domain of human CNNM4 prevent its dimerization. An analogous mutation in mouse CNNM2 impairs its activity in a cellular assay of Mg2+ transport.

Clinical application and progression of monoclonal antibodies targeting HER2 extracellular domain Ⅳ in breast cancer / 中国药房

Human epidermal growth factor receptor 2 （HER2）-positive breast cancer is aggressive and prone to metastasis，and the applications of HER2 agents have improved the prognosis of patients with HER2-positive breast cancer. Among the marketed HER2 agents，macromolecular monoclonal antibodies that target the extracellular domain Ⅳ of HER2 were the cornerstone drugs of HER2-positive breast cancer，including trastuzumab，inetetamab，and margetuximab. Trastuzumab is available for the full-line treatment of breast cancer with sufficient proof of evidence-based medicine，sufficient practical experience and controllable safety. Inetetamab and trastuzumab have similar efficacy and controllable safety in HER2-positive metastatic breast cancer and neoadjuvant/ adjuvant therapy. Margetuximab focuses on patients carrying the CD16A-158F allele，and is an option of posterior line treatment for advanced breast cancer. It is necessary to select the most suitable drugs clinically according to the specific condition of the patient.

Structure of WNT inhibitor adenomatosis polyposis coli down-regulated 1 (APCDD1), a cell-surface lipid-binding protein.

Diverse extracellular proteins negatively regulate WNT signaling. One such regulator is adenomatosis polyposis coli down-regulated 1 (APCDD1), a conserved single-span transmembrane protein. In response to WNT signaling in a variety of tissues, APCDD1 transcripts are highly up-regulated. We have determined the three-dimensional structure of the extracellular domain of APCDD1, and this structure reveals an unusual architecture consisting of two closely apposed ß-barrel domains (ABD1 and ABD2). ABD2, but not ABD1, has a large hydrophobic pocket that accommodates a bound lipid. The APCDD1 ECD can also bind to WNT7A, presumably via its covalently bound palmitoleate, a modification that is common to all WNTs and is essential for signaling. This work suggests that APCDD1 functions as a negative feedback regulator by titrating WNT ligands at the surface of responding cells.

Molecular profile of poorly cohesive gastric carcinoma with special reference to survival.

BACKGROUND: Patients with poorly cohesive gastric carcinoma (PCC) are known to have poor survival. However, detailed molecular biology of PCC has not been elucidated, except for mutations in CDH1 and RHOA. Additionally, the molecular profiles of signet-ring cell carcinoma (SRC) have not been fully investigated. We aimed to investigate the association between molecular profiles and survival in PCC and PCC subtypes. METHODS: The present study included 455 patients with gastric adenocarcinoma underwent radical gastrectomy. Whole-exome sequencing and gene expression profiling were conducted. Patients were classified according to the WHO classification as PCC or non-PCC, with PCC being further classified into SRC, combined, and PCC not-otherwise-specified (NOS). Clinicopathological factors and survival were compared with molecular profiles. RESULTS: Of the patients, 159 were classified with PCC, while 296 were classified with non-PCC. Among PCC, 44 were classified with SRC, 64 with combined, and 51 with PCC-NOS. Mutations in CDH1 and RHOA were remarkably more frequent in PCC than in non-PCC. PCC had worse overall survival (OS) and disease-specific survival (DSS) compared to non-PCC. For PCC, the SRC group had good OS and DSS, whereas PCC-NOS classification with CDH1 mutations was associated with extremely poor survival. In the PCC-NOS and combined groups, patients with mutations in the extracellular domain 1 of CDH1 had poor survival. CONCLUSIONS: Our findings suggest that PCC has poorer survival than non-PCC. Accumulation of CDH1 and RHOA mutations are unique profiles in PCC. Among PCC, CDH1 mutations may play a crucial role in the survival of non-SRC PCC.

Searching for Osmosensing Determinants in Poplar Histidine-Aspartate Kinases.

Previous works have shown the existence of protein partnership, belonging to a MultiStep Phosphorelay (MSP), potentially involved in osmosensing in Populus. The first actor of this signalling pathway belongs to the histidine-aspartate kinase (HK) family, which also includes the yeast osmosensor Sln1, as well as the Arabidopsis putative osmosensor AHK1. In poplar, the homologous AHK1 protein corresponds to a pair of paralogous proteins, HK1a and HK1b, exhibiting an extracellular domain (ECD), as in Sln1 and AHK1. An ECD alignment of AHK1-like proteins, from different plant species, showed a particularly well conserved ECD and revealed the presence of a cache domain. This level of conservation suggested a functional role of this domain in osmosensing. Thus, we tested this possibility by modelling assisted mutational analysis of the cache domain of the Populus HK1 proteins. The mutants were assessed for their ability to respond to different osmotic stress and the results point to an involvement of this domain in HK1 functionality. Furthermore, since HK1b was shown to respond better to stress than HK1a, these two receptors constituted a good system to search for osmosensing determinants responsible for this difference in efficiency. With domain swapping experiments, we finally demonstrated that the cache domain, as well as the second transmembrane domain, are involved in the osmosensing efficiency of these receptors.

Duration of SARS-CoV-2 Serum-Neutralizing Antibodies Against SARS-CoV-2 Dependent on the Individual.

We investigated the persistence of SARS-CoV-2-specific neutralizing antibodies in serum (CoV-2-SNAb) against the "WH-Human 1" coronavirus in 57 convalescent persons from January 2020 to January 2021. The CoV-2-SNAb response against authentic "WH-Human 1" showed a significant (p < 0.01) neutralizing high effect (≥95%) in the following manner: by 94.7% neutralization for up to 6 months, by 73.1% for up to 8 months, and by 31.7% for up to 10 months in correlation with a significant decrease in the concentration of the virus determined by SARS-CoV-2 spike protein extracellular domain and spike-receptor-binding domain (S-RBD). There was neutralizing effect (<95%) when the S-RBD optical density (OD) value was more than 1.0, showing a suitable threshold of S-RBD = 1.0 (antibody-tittering, OD). However, in some convalescent persons, no neutralizing effect (<95%) was observed although the SARS-CoV-2-specific neutralizing antibodies were bound to the S-RBD (OD >1.0). The neutralization of the virus in these cases may not involve S-RBD, but rather B- and T cell memory responses in overall immunity, using the threshold value (OD = 1.0) of S-RBD as a simple and effective method to determine the neutralization effect of the antibody efficacy and use of vaccination in combination with a standard pseudovirus neutralizing assay. We suggest that convalescent persons should contact their physicians 6-month postinfection to test the function of their serum neutralizing antibodies and determine whether administering a SARS-CoV-2 vaccine is necessary to prevent the development of severe illness in the future.

Cloning, expression, purification, and immunoblotting analysis of recombinant type III fibronectin domains of human oncostatin M receptor.

BACKGROUND: The human oncostatin M receptor subunit , commonly known as the oncostatin M receptor (OSMR), is a cell surface protein and belongs to the family of type I cytokine receptors. It is highly expressed in several cancers and is a potential therapeutic target. Structurally, OSMR consists of three major domains: the extracellular, transmembrane, and cytoplasmic domains. The extracellular domain further comprises four Type III fibronectin subdomains. The functional relevance of these type III fibronectin domains is not known yet, and it is of great interest to us to understand their role in OSMR-mediated interactions with other oncogenic proteins. METHODS & RESULTS: The four type III fibronectin domains of hOSMR were amplified by PCR using the pUNO1-hOSMR construct as a template. The molecular size of the amplified products was confirmed by agarose gel electrophoresis. The amplicons were then cloned into a pGEX4T3 vector containing GST as an N-terminal tag. Positive clones with domain inserts were identified by restriction digestion and overexpressed in E. coli Rosetta (DE3) cells. The optimum conditions for overexpression were found to be 1 mM IPTG and an incubation temperature of 37 °C. The overexpression of the fibronectin domains was confirmed by SDS-PAGE, and they are affinity purified by using glutathione agarose beads in three repetitive steps. The purity of the isolated domains analyzed by SDS-PAGE and western blotting showed that they were exactly at their corresponding molecular weights as a single distinct band. CONCLUSION: In this study, we have successfully cloned, expressed, and purified four Type III fibronectin subdomains of hOSMR.

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation.

Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor's glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera.

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer.

BACKGROUND: Precision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism. METHODS: We generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient's cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug's efficacies against HER2 E401G. RESULTS: We confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective. CONCLUSIONS: Afatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.

Extracellular intersubunit interactions modulate epithelial Na+ channel gating.

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the ß-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking ßE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging ßE499C to αN510C or αQ441C altered the Na+ self-inhibition response via distinct mechanisms. Similar to bridging ßE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.

A rapid approach to capture the potential bioactive compounds from Rhizoma Drynariae, utilizing disease-associated mutation in calcium sensing receptor to alter the binding affinity for agonists.

Rhizoma Drynariae (RD) was used clinically to treat osteoporosis in China due to stimulating bone formation and inhibiting bone resorption, however, the bioactive constituents with the dual effect on bone are still unknown exactly. Disease-causing mutations in calcium sensing receptor (CaSR) can alter parathyroid hormone secretion and affect Ca2+ release from bone and Ca2+ reabsorption from kidney, which gives an indication that CaSR is a potential target for developing therapeutics to manage osteoporosis. Herein, a chromatographic approach was established, by immobilizing the mutant CaSR onto the surface of silica gels as stationary phase in a one-step procedure and then adding the different amino acids into mobile phase as competitors, for exploring the binding features of the known agonists and further screening ligands from RD. The mutant CaSR-coated column was prepared rapidly without the complicated purification and separation of the receptor, which had the large capacity of 13.1 mg CaSR /g silica gels and kept a good stability and specificity for at least 35 days. The CaSR mutation can weaken the binding affinities for three agonists, and the largest decreases occurred on the mutational site Thr151Met for neomycin, on the two sites of Asn118Lys and Glu191Lys for gentamicin-C, and on the site Phe612Ser for kanamycin, which gained new insights into their structure-function relationship. The potential bioactive compounds from RD were screened using the mutant CaSR-coated column and were recognized as coumaric acid 4-O-ß-D-glucopyranoside, caffeic acid, and naringin using UPLC-MS. Among them, naringin targeting CaSR gives a possible explanation that RD could manage osteoporosis. These results indicated that, such a rapid and simple method, utilizing disease-associated mutation in CaSR to alter the binding affinity for agonists, can be applied in capturing the potential bioactive compounds efficiently from complex matrices like herb medicines.

Investigation of [3H]diazepam derivatives as allosteric modulators of GABAA receptor α1ß2γ2 subtypes: combination of molecular docking/dynamic simulations, pharmacokinetics/drug-likeness prediction, and QSAR analysis.

In this paper, a data set of [3H] diazepam derivatives was analyzed using various computational methods: molecular docking/dynamic simulations, and QSAR analysis. The main aims of these studies are to understand the binding mechanisms by which benzodiazepines allosterically modulate GABAA receptor α1ß2γ2 subtypes, from inducing neuronal inhibition at lower doses to the anesthetic effect at higher doses, and also, to define the structural requirements that contribute to improving the response of GABAA/α1ß2γ2 receptor to benzodiazepine drugs. The results of the molecular docking study allowed selecting Ro12-6377 and proflazepam as the best modulators for the four binding sites simultaneously. Subsequently, the stability of the selected complexes was investigated by performing molecular dynamics simulation. The latter confirmed the features of both modulators to exert direct effects on the chloride-channel lining residues. Pharmacokinetics and drug-likeness profile were assessed through in silico tool. Furthermore, a QSAR analysis was conducted using an improved vemolecular dynamics simulations proposed byrsion of PLS regression. The goodness of fit and the predictive power of the resulting PLS model were estimated according to internal and external validation parameters: R2 = 0.632, R2adj = 0.584, F = 12.806; p-value = 6.2050e - 07, Q2loo = 0.639, and Q2F3 = 0.813. Clearly, the obtained results ensure the predictive ability of the developed QSAR model for the design of new high-potency benzodiazepine drugs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02029-4.

Proteolytic ectodomain shedding of muscle-specific tyrosine kinase in myasthenia gravis.

Autoantibodies to muscle-specific tyrosine kinase (MuSK) proteins at the neuromuscular junction (NMJ) cause refractory generalized myasthenia gravis (MG) with dyspnea more frequently than other MG subtypes. However, the mechanisms via which MuSK, a membrane protein locally expressed on the NMJ of skeletal muscle, is supplied to the immune system as an autoantigen remains unknown. Here, we identified MuSK in both mouse and human serum, with the amount of MuSK dramatically increasing in mice with motor nerve denervation and in MG model mice. Peptide analysis by liquid chromatography-tandem-mass spectrometry (LC-MS/MS) confirmed the presence of MuSK in both human and mouse serum. Furthermore, some patients with MG have significantly higher amounts of MuSK in serum than healthy controls. Our results indicated that the secretion of MuSK proteins from muscles into the bloodstream was induced by ectodomain shedding triggered by neuromuscular junction failure. The results may explain why MuSK-MG is refractory to treatments and causes rapid muscle atrophy in some patients due to the denervation associated with Ab-induced disruption of neuromuscular transmission at the NMJ. Such discoveries pave the way for new MG treatments, and MuSK may be used as a biomarker for other neuromuscular diseases in preclinical studies, clinical diagnostics, therapeutics, and drug discovery.