Spatially resolved profiling of protein conformation and interactions by biocompatible chemical cross-linking in living cells.

Unlocking the intricacies of protein structures and interactions within the dynamic landscape of subcellular organelles presents a significant challenge. To address this, we introduce SPACX, a method for spatially resolved protein complex profiling via biocompatible chemical cross(x)-linking with subcellular isolation, designed to monitor protein conformation, interactions, and translocation in living cells. By rapidly capturing protein complexes in their native physiological state and efficiently enriching cross-linked peptides, SPACX allows comprehensive analysis of the protein interactome within living cells. Leveraging structure refinement with cross-linking restraints, we identify subcellular-specific conformation heterogeneity of PTEN, revealing dynamic differences in its dual specificity domains between the nucleus and cytoplasm. Furthermore, by discerning conformational disparities, we identify 83 cytoplasm-exclusive and 109 nucleus-exclusive PTEN-interacting proteins, each associated with distinct biological functions. Upon induction of ubiquitin-proteasome system stress, we observe dynamic alterations in PTEN assembly and its interacting partners during translocation. These changes, including the identification of components and interaction sites, are characterized using the SPACX approach. Notably, SPACX enables identification of unique interacting proteins specific to PTEN isoforms, including PTEN and PTEN-Long, through the determination of sequence-specific cross-linking interfaces. These findings underscore the potential of SPACX to elucidate the functional diversity of proteins within distinct subcellular sociology.

Repression of mRNA translation initiation by GIGYF1 via disrupting the eIF3-eIF4G1 interaction.

Viruses can selectively repress the translation of mRNAs involved in the antiviral response. RNA viruses exploit the Grb10-interacting GYF (glycine-tyrosine-phenylalanine) proteins 2 (GIGYF2) and eukaryotic translation initiation factor 4E (eIF4E) homologous protein 4EHP to selectively repress the translation of transcripts such as Ifnb1, which encodes the antiviral cytokine interferon-ß (IFN-ß). Herein, we reveal that GIGYF1, a paralog of GIGYF2, robustly represses cellular mRNA translation through a distinct 4EHP-independent mechanism. Upon recruitment to a target mRNA, GIGYF1 binds to subunits of eukaryotic translation initiation factor 3 (eIF3) at the eIF3-eIF4G1 interaction interface. This interaction disrupts the eIF3 binding to eIF4G1, resulting in transcript-specific translational repression. Depletion of GIGYF1 induces a robust immune response by derepressing IFN-ß production. Our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism has profound implications for the host response to viral infections.

Simultaneous Analysis of Nicarbazin, Diclazuril, Toltrazuril, and Its Two Metabolites in Chicken Muscle and Eggs by In-Syringe Dispersive Solid-Phase Filter Clean-Up Followed by Liquid Chromatography-Tandem Mass Spectrometry.

Nicarbazin (NICA) and triazine anticoccidial drugs (diclazuril (DIZ) and toltrazuril (TOZ)) are the primary strategy for preventing and treating coccidiosis. To prevent the development of drug resistance and mitigate the potential chronic toxicity to humans resulting from prolonged exposure, a liquid chromatography-tandem mass spectrometry method with high reliability and sensitivity was developed to determine NICA, DIZ, TOZ, and its two metabolites in chicken muscle and eggs. Upon establishing the extraction conditions involving 10 mL of acetonitrile and 10 min of sonication, in-syringe dispersive solid-phase extraction with silica was performed in combination with n-hexane clean-up. The selection of isotope peaks of precursor ions and low-mass range scanning allowed the two transitions for the quantification of all compounds. The limits of detection for DIZ and NICA were both 0.1 µg/kg, and for TOZ and metabolites, they were 0.3 µg/kg; the limits of quantitation were 0.3 and 1 µg/kg, respectively. The linear range was 0.25-50 ng/mL with a correlation coefficient r > 0.999. The average recoveries at three spiking levels in muscle and eggs were 90.1-105.2% and 94.0-103.7% with the relative standard deviations of 3.0-8.1% and 3.1-14.4%, respectively. The precision, accuracy, and stability were evaluated by three quality control samples.

Decoding Protein Dynamics in Cells Using Chemical Cross-Linking and Hierarchical Analysis.

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins within cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells.

In vivo cross-linking-based affinity purification and mass spectrometry for targeting intracellular protein-protein interactions.

Comprehensive interactome analysis of targeted proteins is important to understand how proteins work together in regulating functions. Commonly, affinity purification followed by mass spectrometry (AP-MS) has been recognized as the most often used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, which are responsible for key roles in regulation, are easily broken during cell lysis and purification through an AP approach. Herein, we have developed an approach termed in vivo cross-linking-based affinity purification and mass spectrometry (ICAP-MS). By this method, in vivo cross-linking was introduced to covalently fix intracellular PPIs in their functional states to assure all PPIs could be integrally maintained during cell disruption. In addition, the chemically cleavable crosslinkers which were employed enabled unbinding of PPIs for in-depth identification of components within the interactome and biological analysis, while allowing binding of PPIs for cross-linking-mass spectrometry (CXMS)-based direct interaction determination. Multi-level information on targeted PPIs network can be obtained by ICAP-MS, including composition of interacting proteins, as well as direct interacting partners and binding sites. As a proof of concept, the interactome of MAPK3 from 293A cells was profiled with 6.15-fold improvement in identification than by conventional AP-MS. Meanwhile, 184 cross-link site pairs of these PPIs were experimentally identified by CXMS. Furthermore, ICAP-MS was applied in the temporal profiling of MAPK3 interactions under activation by cAMP-mediated pathway. The regulatory manner of MAPK pathways was presented through the quantitative changes of MAPK3 and its interacting proteins at different time points after activation. Therefore, all reported results demonstrated that the ICAP-MS approach may provide comprehensive information on interactome of targeted protein for functional exploration.

"Off-the-Shelf" Allogeneic CAR Cell Therapy-Neglected HvG Effect.

OPINION STATEMENT: Chimeric antigen receptor (CAR) cell therapy offers patients with hematological malignancies a new therapeutic option. Traditionally, autologous T cells are used to generate CAR designed T cells for each patient. However, this method has several drawbacks, the development of allogeneic CAR cell therapy would be a promising breakthrough that could address several of these limitations. From the clinical trials that have published data, the efficacy of allogeneic CAR cell therapy did not meet the expectations. Because of the host-versus-graft (HvG) effect, allogeneic CAR cells are eliminated by the host, resulting in short-term persistence of allogeneic CAR cells and poor efficacy. It is critical to solve the HvG effect of allogeneic CAR cells. The current commonly used methods are suppressing the host's immune system, using HLA-matched homozygous donors, reducing the expression of HLA, targeting alloreactive lymphocytes and eliminating anti-CAR activities. In this review, we will focus on the HvG effect of the "off-the-shelf" allogeneic CAR cell therapy, especially its mechanism and current methods to solve this problem and summarize relevant clinical trial data.

Enhanced protein-protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment.

Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein-protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.

Suborganelle-Specific Protein Complex Analysis Enabled by in Vivo Cross-Linking Coupled with Proximal Labeling.

The identification of the structure of protein complexes in the subcellular niche of cells is necessary to understand their diverse functions. In this study, we developed a suborganelle proteome labeling assisted in vivo cross-linking (SubPiXL) strategy to identify regional protein conformations and interactions in living cells. Due to the mitochondria's functional importance and well-defined compartmental partitions, the specific conformations and interactome of protein complexes located in the mitochondrial matrix were identified. Compared to the commonly used approach of organelle isolation followed by intact mitochondria cross-linking, our method achieved a more refined spatial characterization for the subcompartment of the cellular organelle. Additionally, this approach avoided cross-contamination and cell microenvironment disruption during organelle isolation. As such, we achieved 73% selectivity for mitochondria and 98% specificity of known suborganelle annotation for the mitochondrial matrix and accessible inner membrane. Meanwhile, more protein-protein interactions (PPIs) with high dynamics were captured, resulting in a 1.67-fold increase in the number of PPI identifications in 1/11th of the time. On the basis of these structural cross-links and the specific characterization of the interactome and conformation, the structural dynamics targeted in the mitochondrial matrix were delineated. Mitochondrial matrix-restricted information for proteins with multisubcellular localizations was then clarified. In summary, SubPiXL is a promising technique for the investigation of suborganelle-resolved protein conformation and interaction analysis and contributes to a better understanding of structure-derived functions.

Bibliometric Analyses of Web of Science Illuminate Research Advances of Neuropterida.

Neuropterida is a relatively primitive group of Holometabola. There are about 6500 extant species. Many species of this group are natural enemies and can prey on a variety of agricultural pests. In order to understand the leading research institutions, researchers and research contents, and to predict the future research directions of Neuropterida, the Web of Science core database, from January 1995 to September 2021, was searched with the theme of "Neuropterida or Neuroptera or Megaloptera or Raphidioptera or Lacewing". The results showed that the United States and China published relatively more publications than other countries. In addition, researchers from these two countries had more cooperation with other countries. China Agricultural University ranked the highest in the number of publications and centrality in this field. In addition, it was found that the early research focused on the biological control of Neuropterida by analyzing the keyword burst, whereas the more recent research focused on the phylogeny of Neuropterida. As the first representative chromosome-level genome of Neuropterida has been published, the future research of Neuropterida will focus on the genomic studies and molecular mechanisms of their morphological characters, behavior, historical evolution and so on.

Highly selective enrichment of surface proteins from living cells by photo-crosslinking probe enabled in-depth analysis of surfaceome.

Cell surface-exposed proteins (CSPs), termed the surfaceome, play a key role in many cellular processes. In-depth CSP analysis is significant for screening candidate biomarkers and drug targets. Highly selective enrichment of CSPs in physiological cellular environments is attractive but remains technically challenging. Here, we present a photocrosslinking-assisted cell surface protein enrichment (PCSPE) strategy. In this strategy, CSP labeling would be achieved within 2 min of UV irradiation by developing a new photocrosslinking probe (SDB) followed by one-step enrichment. The enrichment selectivity of CSPs reached 70.5%, and we identified up to 1017 CSPs from living HEK-293T cells, attributed to the high photocrosslinking reactivity and inherent impermeability of SDB, as well as the high cell viability maintained after rapid cell surface labeling to decrease the interference of intracellular proteins. Finally, this strategy was successfully applied to sorafenib-resistance cells for quantitative surfaceome analysis. All results demonstrated that our developed PCSPE method might provide a valuable toolkit for in-depth surfaceome profiling and comprehensive functional analysis.

[Mirror cutting-assisted orthogonal digestion enabling large-scale and accurate protein complex characterization].

Protein complexes are involved in a variety of biological activities. Accurate and comprehensive characterization of the structures and interactions of protein complexes is crucial in determining their biological functions. Chemical cross-linking coupled with mass spectrometry (CXMS) is an emerging investigative technique for protein complexes. CXMS enables the sensitive high-throughput analysis of protein complexes without the requirements of molecular weight and purification. These attributes have spurred the increased use of CXMS for the structure and interaction characterization of purified protein complexes and complicated cell lysate samples. CXMS utilizes chemical cross-linking reagents to covalently connect two reactive amino acids in or between proteins that are spatially close to each other. Subsequently, the cross-linked proteins are digested into cross-linked peptides, followed by LC-MS/MS analysis, as well as database searching to provide cross-linking information for the composition, interaction, and structural site distance restrictions of protein complex identification. Therefore, identification of cross-linked sites has a decisive influence on the characterization of protein complexes. This identification is limited by the unsatisfactory quality of the cross-linked peptide spectrum. Insufficient b/y fragment ions and poor continuity of amino acid sequence matching lead to low coverage and accuracy of cross-linked site identification. Based on the complementary feature of mirror-cutting digestion, an orthogonal digestion strategy based on LysargiNase combined with trypsin was developed in this study. Trypsin is the most commonly utilized digestion enzyme in proteomics, with extremely high enzyme activity and specificity. Trypsin generates C-terminally charged peptides after lysine (K) and arginine (R). LysargiNase is a mirror protease complementary to trypsin that cleaves before the K and R residues. This generates peptides with an N-terminal positively-charged residue. Owing to the different physical and chemical micro-environments of the cross-linked peptides digested by LysargiNase and trypsin, the behavior of their detection ability in MS analysis is diverse. Using the orthogonal digestion strategy, both simple and complicated cross-linked samples were analyzed in this study. For the analysis of bovine serum albumin (BSA), 291 pairs of non-redundant cross-linked sites were obtained, of which 216 pairs of cross-linked sites were provided by trypsin digestion, whereas 75 pairs of cross-linked sites were exclusively supplied by LysargiNase digestion. Except for the 35% increase in the number of identified cross-linked sites, 32% of the spectra of the commonly identified cross-linked peptides have better quality with more b-type fragment ions and consecutive sequence matching. Furthermore, for the Escherichia coli sample, 726 pairs of cross-linked sites were obtained in total, among which, 624 and 274 pairs were identified from trypsin and LysargiNase digestion, respectively. LysargiNase digestion yielded 120 individual cross-linked sites, which resulted in a 16% increase in single trypsin digestion. Consistent with the BSA sample, the quality was improved in 35% of the spectra of commonly identified cross-linked peptides. Corresponding to the identified cross-linked peptides, 242 structural constraints with 607 pairs of intra-cross-linked sites and 29 sets of protein-protein interactions with 119 pairs of inter-cross-linked sites were obtained. The collective results demonstrated that, mirror cutting-assisted orthogonal digestion strategy could significantly increase the number of identified fragment ions and amino acid sequences matching the continuity of the spectra by contributing b-and y-type ions, respectively. This improved the accuracy and coverage of cross-linked peptide identification. The findings additionally demonstrate the superiority of our method in the accurate identification of the cross-linked peptide spectra and the increased number of identified cross-linked sites. In a word, this method is expected to provide new insights for the large-scale and highly accurate characterization of protein complexes.

Selective Removal of Unhydrolyzed Monolinked Peptides from Enriched Crosslinked Peptides To Improve the Coverage of Protein Complex Analysis.

Chemical crosslinking combined with mass spectrometry (CXMS) has allowed the global characterization of protein complexes with high throughput and accuracy. Although enrichable crosslinkers have been introduced to exclude the interference of regular peptides, the crosslinked peptide identification is still severely inhibited by a large amount of monolinked peptides. In this work, we proposed a strategy called MoTE (unhydrolyzed Monolinked peptide Targeting Elimination) to remove the unhydrolyzed monolinked peptides, while enriching crosslinked peptides for regular peptide removal. In this strategy, followed by the crosslinking reaction, an amine biotin reagent was used to block the unreacted reactive groups on the crosslinker, and subsequently, the crosslinked proteins were tagged by a cleavable biotin-azide ligand based on click chemistry for enrichment. The following crosslinked protein digestion, purification by streptavidin beads, and release by chemical cleavage of the biotin-azide ligand were sequentially performed. In this case, the amine biotin-blocked unhydrolyzed monolinked peptides with the unbreakable arm remained on the streptavidin beads, which realized selective removal without any additional steps. By combining in vivo crosslinking with our proposed MoTE strategy for protein complex analysis of the HeLa cell, the number of high reliability (score

The first mitochondrial genome for Phaudidae (Lepidoptera) with phylogenetic analyses of Zygaenoidea.

Phauda flammans Walker belongs to Phaudidae (Lepidoptera), which is a holometabolous and leaf-eating pest that harms trees. So far, there is no mitochondrial (mt) genome reported of Phaudidae. Herein, we sequenced and annotated the complete mt genome of P. flammans representing the first mt genome of Phaudidae and predicted the secondary structures of its RNAs in this study. This mt genome is 15470 bp long consisting of 13 protein coding genes (PCGs), 22 tRNAs, 2 rRNAs and the control region, which are usually conserved in insects. Most PCGs used the standard ATN start codons and complete TAA/TAG termination codons. Almost all of tRNA genes exhibited cloverleaf secondary structures except that the dihydorouridine (DHU) arm of tRNASer(AGN) was absent. The phylogenetic analyses using both Bayesian inference (BI) and maximum likelihood (ML) methods all supported that Phaudidae was a single family being the sister group to Zygaenidae. More mt genomes are needed to better understand the phylogenetic relationships within Zygaenoidea in the future.

The complete mitochondrial genome of Hemerobius simulans Walker (Neuroptera, Hemerobiidae).

The complete mitochondrial (mt) genome of Hemerobius simulans Walker (Neuroptera, Hemerobiidae) is reported in this work. The whole mt genome is 17,985 bp long and contains 37canonical genes and an A + T-rich region, which is the same with insect ancestral mt genome arrangement. All 13 PCGs used the typical ATN as initiation codons. The control region of H. simulans mt genome is 1,416 bp long and the base composition is 90.0% of A + T. The phylogenetic analysis revealed that Hemerobiidae was monophyletic and was the sister group to Chrysopidae.

Reliable Analysis of the Interaction between Specific Ligands and Immobilized Beta-2-Adrenoceptor by Adsorption Energy Distribution.

Although a comparatively robust method, immobilized protein-based techniques have displayed limited precision and inconsistent results due to a lack of strategy for the accurate selection of drug adsorption models on the protein surface. We generated the adsorption data of three drugs on immobilized beta-2-adrenoceptor (ß2-AR) by frontal affinity chromatography-mass spectrometry (FAC-MS) and site-specific competitive FAC-MS. Using adsorption energy distribution (AED) calculations, we achieved the best adsorption models for the binding of salbutamol, terbutaline, and pseudoephedrine to immobilized ß2-AR. The Langmuir model proved to be desirable for describing the adsorptions of salbutamol and terbutaline on immobilized ß2-AR, while the bi-Langmuir model was favorable to characterize the adsorption of pseudoephedrine on the receptor. Relying on the accurate determination of association constants, we presented an efficient approach for ß2-AR ligand screening based on the loss of breakthrough time of an indicator drug caused by the inclusion of competitive drugs in the mobile phase. We concluded that the current strategy enables the reliable and accurate analysis of G protein-coupled receptor (GPCR)-drug interaction. The percentage change in the breakthrough time for drugs can provide useful information for estimating their binding affinity to the receptor. This approach builds a powerful platform for high-throughput ligand screening.

Investigation on temperature-induce conformational change of immobilized ß2 adrenergic receptor.

The ß2 adrenergic receptor (ß2-AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. Purified ß2-AR was immobilized on macroporous silica gel to obtain liquid chromatographic stationary phase. The resulting phase was packed into a stainless steel column (4.6 × 50 mm, 7 µm) and used for on-line chromatographic system. When column oven temperature increased from 20.0 °C to 40.0 °C, uncomplete separate chromatographic peaks of ephedrine and pseudoephedrine as receptor conformational probe were gradually merged into one peak, meanwhile retention time and resolution of the probes were reduced correspondingly, which suggested that temperature could regulate protein conformation. Temperature-induced conformational change of immobilized ß2-AR, especially changes at higher temperatures, indicated that constructed receptor chromatography could simulate fever disease state of human body and clarify receptor conformation change at pathological condition. At the same time this study could also provide new ideas for screening active components in pathological conditions.

Target-directed screening of the bioactive compounds specifically binding to ß2-adrenoceptor in Semen brassicae by high-performance affinity chromatography.

The bioactive ingredients in Semen sinapis were rapidly screened by immobilized ß2-adrenoceptor (ß2-AR) and target-directed molecular docking. The methods involved the attachment of ß2-AR using any amino group in the receptor, the simultaneous separation and identification of the retention compounds by high-performance affinity chromatography; the binding mechanism of the interesting compound to the receptor was investigated by zonal elution and molecular docking. Sinapine in Semen sinapis was proved to be the bioactive compound that specifically binds to the immobilized receptor. The association constant of sinapine to ß2-AR was determined to be 1.36 × 10(5) M(-1) with a value of 1.27 × 10(-6) M for the number of binding sites. Ionic bond was believed to be the driving force during the interaction between sinapine and ß2-AR. It is possible to become a powerful alternative for rapid screening of bioactive compounds from a complex matrix such as traditional Chinese medicine and further investigation on the drug-receptor interaction.

Binding of caffeic acid to human serum albumin by the retention data and frontal analysis.

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high-performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 10(4) /m. The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm. The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug-protein interaction. The proposed model also has the advantages of ligand saving and rapid operation.