Structure-Based Rational and General Strategy for Stabilizing Single-Chain T-Cell Receptors to Enhance Affinity.

The T-cell receptor (TCR) is a crucial molecule in cellular immunity. The single-chain T-cell receptor (scTCR) is a potential format in TCR therapeutics because it eliminates the possibility of αß-TCR mispairing. However, its poor stability and solubility impede the in vitro study and manufacturing of therapeutic applications. In this study, some conserved structural motifs are identified in variable domains regardless of germlines and species. Theoretical analysis helps to identify those unfavored factors and leads to a general strategy for stabilizing scTCRs by substituting residues at exact IMGT positions with beneficial propensities on the consensus sequence of germlines. Several representative scTCRs are displayed to achieve stability optimization and retain comparable binding affinities with the corresponding αß-TCRs in the range of µM to pM. These results demonstrate that our strategies for scTCR engineering are capable of providing the affinity-enhanced and specificity-retained format, which are of great value in facilitating the development of TCR-related therapeutics.

Visual differences in topography-guided versus wavefront-optimized LASIK in the treatment of myopia: a Meta-analysis.

AIM: To investigate the potential differences between topography-guided (TG) and wavefront-optimized (WFO) laser in situ keratomileusis (LASIK) for the treatment of myopia. METHODS: A systematic literature search was performed to determine relevant trials comparing LASIK with TG and WFO from the time of library construction to August 2020, and The PubMed, Cochrane, Web of Science, EMBASE and Chinese databases (i.e. CNKI, CBM, WAN FANG and VIP) were accessed. The data on visual acuity, refractive status and wavefront aberration were retrieved and evaluated from three to six months after surgery. STATA (version 14.0) software was used for statistical analysis. A cumulative Meta-analysis was simultaneously performed. RESULTS: Eleven studies with a total of 1425 eyes were incorporated. No statistically significant differences were evident between TG and WFO ablation in the proportion of eyes achieving an uncorrected distance visual acuity (UCVA) of 20/20 or better (P=0.377), gaining one line or more (P=0.05), postoperative cylinder (P=0.40), vertical coma (P=0.593) and horizontal coma (P=0.957). After TG ablation, the proportion of the patients' eyes of which postoperative refraction is within ±0.5 diopter of the target refraction was significantly higher than that undergoes WFO (P=0.003). As opposed to the WFO group, manifest refraction spherical equivalent (MRSE; P=0.000) was lower, and UCVA (P=0.005) was better in the TG group. The higher-order aberrations (HOAs; P=0.000), spherical aberration (P=0.000) and coma (P=0.000) were significantly lower in TG group. The cumulative Meta-analysis illustrated that the proportion of eyes achieving UCVA of 20/20 or better, postoperative refraction within ±0.5 diopter, and MRSE has steady between the two groups. CONCLUSION: Both TG-LASIK and WFO-LASIK are safe, effective, and predictable for correcting myopia. TG-LASIK may produce fewer aberration and is more precise than WFO-LASIK.

The Application of Nanobody in CAR-T Therapy.

Chimeric antigen receptor (CAR) T therapy represents a form of immune cellular therapy with clinical efficacy and a specific target. A typical chimeric antigen receptor (CAR) construct consists of an antigen binding domain, a transmembrane domain, and a cytoplasmic domain. Nanobodies have been widely applied as the antigen binding domain of CAR-T due to their small size, optimal stability, high affinity, and manufacturing feasibility. The nanobody-based CAR structure has shown a proven function in more than ten different tumor-specific targets. After being transduced in Jurkat cells, natural killer cells, or primary T cells, the resulting nanobody-based CAR-T or CAR-NK cells demonstrate anti-tumor effects both in vitro and in vivo. Interestingly, anti-BCMA CAR-T modulated by a single nanobody or bi-valent nanobody displays comparable clinical effects with that of single-chain variable fragment (scFv)-modulated CAR-T. The application of nanobodies in CAR-T therapy has been well demonstrated from bench to bedside and displays great potential in forming advanced CAR-T for more challenging tasks.

[Screen absorption enhancer for intranasal administration preparations of paeoniflorin based on nasal perfusion method in rats].

This experiment was aimed to screen the absorption enhancer for intranasal administration preparations of paeoniflorin. In this study, HPLC method for determination of paeoniflorin in perfusion liquid was established and the improved model of nasal perfusion in rats was used to screen out the species and amounts of absorption enhancer. In order to avoid the influence of the secretion and absorption of nasal cavity on the volume of perfusion fluid, the residual dose was calculated by using the volume correction method. Linear regression was carried out between the logarithm to the percentage of the residual dose and the corresponding time, and the slope of the regression line was exactly the absorption rate constant. Experimental results showed that hydroxypropyl-ß-cyclodextrin and water-soluble azone can significantly improve the nasal absorption of paeoniflorin. Furthermore, water-soluble azone had the highest absorption rate constant and the best promoting penetration effect on intranasal administration preparations of paeoniflorin. It was also found that when the mass concentration of water-soluble azone in the perfusion liquid increased from 5 g•L⁻¹ to 20 g•L⁻¹, the absorption rate constant was gradually increased and peaked at 20 g•L⁻¹. When the mass concentration was increased to 30 g•L⁻¹, the absorption rate constant was decreased, indicating that the best mass concentration of water-soluble azone was 20 g•L⁻¹.

Prokaryotic and Highly-Repetitive WD40 Proteins: A Systematic Study.

As an ancient protein family, the WD40 repeat proteins often play essential roles in fundamental cellular processes in eukaryotes. Although investigations of eukaryotic WD40 proteins have been frequently reported, prokaryotic ones remain largely uncharacterized. In this paper, we report a systematic analysis of prokaryotic WD40 proteins and detailed comparisons with eukaryotic ones. About 4,000 prokaryotic WD40 proteins have been identified, accounting for 6.5% of all WD40s. While their abundances are less than 0.1% in most prokaryotes, they are enriched in certain species from Cyanobacteria and Planctomycetes, and participate in various functions such as prokaryotic signal transduction and nutrient synthesis. Comparisons show that a higher proportion of prokaryotic WD40s tend to contain multiple WD40 domains and a large number of hydrogen bond networks. The observation that prokaryotic WD40 proteins tend to show high internal sequence identity suggests that a substantial proportion of them (~20%) should be formed by recent or young repeat duplication events. Further studies demonstrate that the very young WD40 proteins, i.e., Highly-Repetitive WD40s, should be of higher stability. Our results have presented a catalogue of prokaryotic WD40 proteins, and have shed light on their evolutionary origins.

Structural insights into Gemin5-guided selection of pre-snRNAs for snRNP assembly.

In cytoplasm, the survival of motor neuron (SMN) complex delivers pre-small nuclear RNAs (pre-snRNAs) to the heptameric Sm ring for the assembly of the ring complex on pre-snRNAs at the conserved Sm site [A(U)4-6G]. Gemin5, a WD40 protein component of the SMN complex, is responsible for recognizing pre-snRNAs. In addition, Gemin5 has been reported to specifically bind to the m7G cap. In this study, we show that the WD40 domain of Gemin5 is both necessary and sufficient for binding the Sm site of pre-snRNAs by isothermal titration calorimetry (ITC) and mutagenesis assays. We further determined the crystal structures of the WD40 domain of Gemin5 in complex with the Sm site or m7G cap of pre-snRNA, which reveal that the WD40 domain of Gemin5 recognizes the Sm site and m7G cap of pre-snRNAs via two distinct binding sites by respective base-specific interactions. In addition, we also uncovered a novel role of Gemin5 in escorting the truncated forms of U1 pre-snRNAs for proper disposal. Overall, the elucidated Gemin5 structures will contribute to a better understanding of Gemin5 in small nuclear ribonucleic protein (snRNP) biogenesis as well as, potentially, other cellular activities.

WDSPdb: a database for WD40-repeat proteins.

WD40-repeat proteins, as one of the largest protein families, often serve as platforms to assemble functional complexes through the hotspot residues on their domain surfaces, and thus play vital roles in many biological processes. Consequently, it is highly required for researchers who study WD40 proteins and protein-protein interactions to obtain structural information of WD40 domains. Systematic identification of WD40-repeat proteins, including prediction of their secondary structures, tertiary structures and potential hotspot residues responsible for protein-protein interactions, may constitute a valuable resource upon this request. To achieve this goal, we developed a specialized database WDSPdb (http://wu.scbb.pkusz.edu.cn/wdsp/) to provide these details of WD40-repeat proteins based on our recently published method WDSP. The WDSPdb contains 63,211 WD40-repeat proteins identified from 3383 species, including most well-known model organisms. To better serve the community, we implemented a user-friendly interactive web interface to browse, search and download the secondary structures, 3D structure models and potential hotspot residues provided by WDSPdb.

[Observation on the effects of the treatment of sillicosis merger asthma].

OBJECTIVE: To investigate Salmeterol/Fluticasone Propionate and Totropiumi treatment of Sillicosis merger Asthma. METHODS: 30 patients with Sillicosis merger Asthma were randomly divided into group Salmeterol/Fluticasone Propionate( Single group) ( n=14) and group Salmeterol/Fluticasone Propionate and Totropiumi (Joint group) ( n= 16), patient in single group were only given Salmeterol/Fluticasone Propionate (50 f.Lg Bid) inhaling,and those in Joint group were given Salmeterol/Fluticasone Propionate (50 f.Lg Bid) and Totropiumi ( 18 f.Lg Qd) inhaling. The treatment was last for 6 months.Before the treatment,evaluation of the two groups of Sillicosis installment,determination their foungation lung function and ACT score .. After the cause of treatment, lung function FEV10/FVC(% ), FEV10 pred%, FEV10(ml), ACT score, the incidence of side effects of two groups were compared and analyzed. RESULT: The two groups before the treatment of lung fuction and ACT score had no statistically significant difference. The two groups after treatment of lung fuction FEV10/FVC (% ),FEV10 pred%, ACT score obviously higher than before treatment (P<0.05), Joint group in FEV1/FVC(% ), ACT score significantly higher than in Single group (?<0.05), Joint group acute attack times(0.98±0.79)/time lower than Single group (2.10 ± 0.81 )/time (t=3.86,P<0.05). There were no significant side effect in two groups. CONCLUSION: Salmeterol/Fluticasone Propionate or the combination of Salmeterol/Fluticasone Propionate and Totropiumi can improve lung function and clinical symptoms of patients with Sillicosis merger Asthma. It is also better that the combination of Salmeterol/Fluticasone Propionate and Totropiumi obviously improve clinical symptoms of patients and reduice acute attack times.

A method for WD40 repeat detection and secondary structure prediction.

WD40-repeat proteins (WD40s), as one of the largest protein families in eukaryotes, play vital roles in assembling protein-protein/DNA/RNA complexes. WD40s fold into similar ß-propeller structures despite diversified sequences. A program WDSP (WD40 repeat protein Structure Predictor) has been developed to accurately identify WD40 repeats and predict their secondary structures. The method is designed specifically for WD40 proteins by incorporating both local residue information and non-local family-specific structural features. It overcomes the problem of highly diversified protein sequences and variable loops. In addition, WDSP achieves a better prediction in identifying multiple WD40-domain proteins by taking the global combination of repeats into consideration. In secondary structure prediction, the average Q3 accuracy of WDSP in jack-knife test reaches 93.7%. A disease related protein LRRK2 was used as a representive example to demonstrate the structure prediction.

Crystal structures of the human histone H4K20 methyltransferases SUV420H1 and SUV420H2.

SUV420H1 and SUV420H2 are two highly homologous enzymes that methylate lysine 20 of histone H4 (H4K20), a mark that has been implicated in transcriptional regulation. In this study, we present the high-resolution crystal structures of human SUV420H1 and SUV420H2 in complex with SAM, and report their substrate specificity. Both methyltransferases have a unique N-terminal domain and Zn-binding post-SET domain, and prefer the monomethylated histone H4K20 as a substrate in vitro. No histone H4K20 trimethylation activity was detected by our radioactivity-based assay for either enzyme, consistent with the presence of a conserved serine residue that forms a hydrogen bond with the target lysine side-chain and limits the methylation level.

Identifying the hotspots on the top faces of WD40-repeat proteins from their primary sequences by ß-bulges and DHSW tetrads.

The analysis of 36 available crystal structures of WD40 repeat proteins reveals widespread existence of a beta-bulge formed at the beginning of strand a and the end of strand b, termed as WD(b-a) bulge: among a total of 259 WD40 blades, there are 243 such ß-bulges. The R(1) positions in these WD(b-a) bulges have fair distributions of Arg, His, Ile, Leu, Lys, Met, Phe, Trp, Tyr and Val residues. These residues protrude on the top face of the WD40 proteins and can serve as hotspots for protein-protein interactions. An analysis of 29 protein complexes formed by 17 WD proteins reveals that these R(1) residues, along with two other residues (R(1)-2 and D-1), are indeed widely involved in protein-protein interactions. Interestingly, these WD(b-a) bulges can be easily identified by the 4-amino acid sequences of (V, L, I), R(1), R(2), (V, L, I), along with some other significant amino acids. Thus, the hotspots of WD40 proteins on the top face can be readily predicted based on the primary sequences of the proteins. The literature-reported mutagenesis studies for Met30, MDV1, Tup11, COP1 and SPA1, which crystal structures are not available, can be readily understood based on the feature-based method. Applying the method, the twelve potential hotspots on the top face of Tup11 from S. japonicas have been identified. Our ITC measurements confirm seven of them, Tyr382, Arg284, Tyr426, Tyr508, Leu559, Lys575 and Ile601, are essential for recognizing Fep1. The ITC measurements further convinced that the feature-based method provides accurate prediction of hotspots on the top face.

The effect of Asp-His-Ser/Thr-Trp tetrad on the thermostability of WD40-repeat proteins.

We recently found that Asp-His-Ser/Thr-Trp hydrogen-bonded tetrads are widely and uniquely present in the WD40-repeat proteins. WDR5 protein is a seven WD40-repeat propeller with five such tetrads. To explore the effect of the tetrad on the structure and stability of WD40-repeat proteins, the wild-type WDR5 and its seven mutants involving the substitutions of tetrad residues have been isolated. The crystal structures of the wild-type WDR5 and its three WDR5 mutants have been determined by X-ray diffraction method. The mutations of the tetrad residues are found not to change the basic structural features. The denaturing profiles of the wild type and the seven mutants with the use of denaturant guanidine hydrochloride have been studied by circular dichroism spectroscopy to determine the folding free energies of these proteins. The folding free energies of the wild type and the S62A, S146A, S188A, D192E, W330F, W330Y, and D324E mutants are measured to be about -11.6, -2.7, -3.1, -2.9, -3.6, -7.1, -7.0, and -7.5 kcal/mol, respectively. These suggest that (1) the hydrogen bonds in these hydrogen bond networks are unusually strong; (2) each hydrogen-bonded tetrad provides over 12 kcal/mol stability to the protein; thus, the removal of any single tetrad would cause unfolding of the protein; (3) since there are five tetrads, the protein must be in a highly unstable state without the tetrads, which might be related to its biological functions.

A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase.

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two-layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn-bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate-determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn-bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate-determining step is the proton transfer from Zn-OH to 6-NH(2) of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate.

Is Asp-His-Ser/Thr-Trp tetrad hydrogen-bond network important to WD40-repeat proteins: a statistical and theoretical study.

WD40-repeat proteins are abundant and play important roles in forming protein complexes. The domain usually has seven WD40 repeats, which folds into a seven beta-sheet propeller with each beta-sheet in a four-strand structure. An analysis of 20 available WD40-repeat proteins in Protein Data Bank reveals that each protein has at least one Asp-His-Ser/Thr-Trp (D-H-S/T-W) hydrogen-bonded tetrad, and some proteins have up to six or seven such tetrads. The relative positions of the four residues in the tetrads are also found to be conserved. A sequence alignment analysis of 560 WD40-repeat protein sequences in human reveals very similar features, indicating that such tetrad may be a general feature of WD40-repeat proteins. We carried out density functional theory and found that these tetrads can lead to significant stabilization including hydrogen-bonding cooperativity. The hydrogen bond involving Trp is significant. These results lead us to propose that the tetrads may be critical to the stability and the mechanism of folding of these proteins.

Catalytic mechanism of SHCHC synthase in the menaquinone biosynthesis of Escherichia coli: identification and mutational analysis of the active site residues.

(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase (MenH) is an alpha/beta fold enzyme containing a catalytically essential serine-histidine-aspartate triad typical of serine proteases but catalyzes a pyruvate elimination reaction initiated by alpha-proton abstraction in the menaquinone biosynthetic pathway of Escherichia coli. In this study, we identify the active site residues in the synthase through sequence analysis and structural modeling and study their mechanistic roles in MenH catalysis. Steady-state kinetic characterization of site-directed mutants of the active site residues shows that three conserved arginine residues (Arg-90, Arg-124, and Arg-168) likely form ionic salt bridges with three carboxylate groups of the substrate in the Michaelis complex and that the side-chain polar groups of the conserved tyrosine (Tyr-85) and tryptophan (Trp-147) residues likely donate hydrogen bonds to form an "oxyanion hole". In addition, the pH dependence of the MenH kinetic properties reveals a catalytic base with a pK(a) highly dependent on the hydroxyl group of the triad serine residue in the enzymatic reaction. Moreover, proton inventory experiments demonstrate that the SHCHC synthase adopts one-proton catalysis like many serine proteases. These results allow the proposal of a mechanism in which the histidine residue of the MenH triad serves as a general base catalyst to deprotonate the triad seryl hydroxyl group in the alpha-proton abstraction from the substrate. As such, the MenH triad performs a simple and fundamental proton transfer reaction occurring repeatedly in the reactions catalyzed by serine proteases and alpha/beta fold hydrolases, suggesting a common evolutionary origin for all serine-histidine-aspartate triads serving different catalytic functions.

Theoretical study of the catalytic mechanism and metal-ion dependence of peptide deformylase.

The reaction pathway of deformylation catalyzed by E. coli peptide deformylase (PDF) has been investigated by the density functional theory method of PBE1PBE on a small model and by a two-layer ONIOM method on a realistic protein model. The deformylation proceeds in sequential steps involving nucleophilic addition of metal-coordinated water/hydroxide to the carbonyl carbon of the formyl group, proton transfer, and cleavage of the C-N bond. The first step is rate-determining for the deformylation, which occurs through a pentacoordinated metal center. The estimated activation energies with the ONIOM method are about 23.0, 15.0, and 14.9 kcal/mol for Zn-, Ni-, and Fe-PDFs, respectively. These calculated barriers are in close agreement with experimental observations. Our results demonstrate that the preference for metal coordination geometry exerts a significant influence on the catalytic activity of PDFs by affecting the activation of the carbonyl group of the substrate, the deprotonation of the metal-coordinated water, and the stabilization of the transition state. This preference for coordination geometry is mainly determined by the ligand environment and the intrinsic electronic structures of the metal center in the active site of the PDFs.

A 4.3 Mb YAC Contig in Human Xp11.2: Long-Range Restriction Mapping and Identification of CpG Islands.

The Xp11.2 region o the human X chromosome contains genes involved in a number of inherited diseased, with at least one locus that escapes X chromosome inactivation, as well as abnormal methylation polymorphism. We isolated a series of yeast artificial chromosome (YAC) clones by hybridization screening with DNA probes localized within this region and assembled them into a 4.3 Mb contig spanning from Xp11.21 to Xp11.23 by a combination of Alu-PCR fingerprinting, STS-PCR and DNA probe cross hybridization. On the basis of these overlapping YAC clones we have constructed the long-range restriction map of this interval and placed exactly some DNA markers. Four CpG-dense regions between ARAF1 and OATL2 were identified based on the long-range restriction mapping, which indicated the distribution of genes within this interval. It should assist in the future nucleic acid sequence analysis and novel gene identification in this region.