kDa Protein Research Articles

Heterologous expression of BACE1 and its interaction with Codonopsis pilosula polysaccharides and Lobetyolin

BACE1, a crucial enzyme in the amyloid-β deposition theory of Alzheimer's disease (AD), is targeted by Codonopsis pilosula, a traditional tonic believed to impede AD onset. However, the specific active compounds responsible for its effects remain elusive. Our prior network pharmacology research identified C. pilosula polysaccharides (CPPS) and Lobetyolin may serve as potential inhibitors of AD by suppressing amyloidogenesis. Here, we recombinantly expressed BACE1 under varied conditions and assessed its activity using Fluorescence Resonance Energy Transfer technology. Through spectroscopy, molecular docking, and dynamics, we elucidated the interactions of CPPS, Lobetyolin, and BACE1. Optimal BACE1 expression occurred at 22 °C with 0.4 mM IPTG for 6 h, yielding a 72 kDa protein. Enzyme kinetics displayed a maximum rate of 4096 μmol/min and a Michaelis constant of 16 mg/mL for BACE1. Spectroscopic analysis revealed differing binding affinities of the compounds at various temperatures, peaking at 293 K. Lobetyolin exhibited superior binding to BACE1 compared to CPPS, driven by hydrophobic and electrostatic forces. Molecular docking and dynamics highlighted hydrophobic amino acids' role in BACE1 interactions with Lobetyolin and CPPS, with binding energy < −1.2 kcal/mol signifying strong affinities. Notably, Lobetyolin and CPPS showed higher BACE1 affinity than APP, with the Lobetyolin-BACE1 complex being the most stable.

The Role of Intestinal Cytochrome P450s in Vitamin D Metabolism.

Vitamin D hydroxylation in the liver/kidney results in conversion to its physiologically active form of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 controls gene expression through the nuclear vitamin D receptor (VDR) mainly expressed in intestinal epithelial cells. Cytochrome P450 (CYP) 24A1 is a catabolic enzyme expressed in the kidneys. Interestingly, a recently identified mutation in another CYP enzyme, CYP3A4 (gain-of-function), caused type III vitamin D-dependent rickets. CYP3A are also expressed in the intestine, but their hydroxylation activities towards vitamin D substrates are unknown. We evaluated CYP3A or CYP24A1 activities on vitamin D action in cultured cells. In addition, we examined the expression level and regulation of CYP enzymes in intestines from mice. The expression of CYP3A or CYP24A1 significantly reduced 1,25(OH)2D3-VDRE activity. Moreover, in mice, Cyp24a1 mRNA was significantly induced by 1,25(OH)2D3 in the intestine, but a mature form (approximately 55 kDa protein) was also expressed in mitochondria and induced by 1,25(OH)2D3, and this mitochondrial enzyme appears to hydroxylate 25OHD3 to 24,25(OH)2D3. Thus, CYP3A or CYP24A1 could locally attenuate 25OHD3 or 1,25(OH)2D3 action, and we suggest the small intestine is both a vitamin D target tissue, as well as a newly recognized vitamin D-metabolizing tissue.

Dysregulation of SNX1-retromer axis in pharmacogenetic models of Parkinson’s disease

Since the identification of vacuolar protein sorting (VPS) 35, as a causative molecule for familial Parkinson’s disease (PD), retromer-mediated endosomal machinery has been a rising factor in the pathogenesis of the disease. The retromer complex cooperates with sorting nexin (SNX) dimer and DNAJC13, another causal molecule in PD, to transport cargoes from endosomes to the trans-Golgi network, and is also involved in mitochondrial dynamics and autophagy. Retromer dysfunction may induce neuronal death leading to PD via several biological cascades, including misfolded, insoluble α-synuclein (aS) accumulation and mitochondrial dysfunction; however, the detailed mechanisms remain poorly understood. In this study, we showed that the stagnation of retromer-mediated retrograde transport consistently occurs in different PD-mimetic conditions, i.e., overexpression of PD-linked mutant DNAJC13, excess aS induction, or toxin-induced mitochondrial dysfunction. Mechanistically, DNAJC13 was found to be involved in clathrin-dependent retromer transport as a functional modulator of SNX1 together with heat shock cognate 70 kDa protein (Hsc70), which was controlled by the binding and dissociation of DNAJC13 and SNX1 in an Hsc70 activity-dependent manner. In addition, excess amount of aS decreased the interaction between SNX1 and VPS35, the core component of retromer. Furthermore, R33, a pharmacological retromer chaperone, reduced insoluble aS and mitigated rotenone-induced neuronal apoptosis. These findings suggest that retrograde transport regulated by SNX1-retromer may be profoundly involved in the pathogenesis of PD and is a potential target for disease-modifying therapy for the disease.

The CryoEM structure of human serum albumin in complex with ligands

Human serum albumin (HSA) is the most prevalent plasma protein in the human body, accounting for 60 % of the total plasma protein. HSA plays a major pharmacokinetic function, serving as a facilitator in the distribution of endobiotics and xenobiotics within the organism. In this paper we report the cryoEM structures of HSA in the apo form and in complex with two ligands (salicylic acid and teniposide) at a resolution of 3.5, 3.7 and 3.4 Å, respectively. We expand upon previously published work and further demonstrate that sub-4 Å maps of ∼60 kDa proteins can be routinely obtained using a 200 kV microscope, employing standard workflows. Most importantly, these maps allowed for the identification of small molecule ligands, emphasizing the practical applicability of this methodology and providing a starting point for subsequent computational modeling and in silico optimization.

Multiple transcription factors involved in the response of Chinese cabbage against Plasmodiophora brassicae.

Clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae, leads to the formation of galls, commonly known as pathogen-induced tumors, on the roots of infected plants. The identification of crucial regulators of host tumor formation is essential to unravel the mechanisms underlying the proliferation and differentiation of P. brassicae within plant cells. To gain insight into this process, transcriptomic analysis was conducted to identify key genes associated with both primary and secondary infection of P. brassicae in Chinese cabbage. Our results demonstrate that the k-means clustering of subclass 1, which exhibited specific trends, was closely linked to the infection process of P. brassicae. Of the 1610 differentially expressed genes (DEGs) annotated in subclass 1, 782 were identified as transcription factors belonging to 49 transcription factor families, including bHLH, B3, NAC, MYB_related, WRKY, bZIP, C2H2, and ERF. In the primary infection, several genes, including the predicted Brassica rapa probable pectate lyase, RPM1-interacting protein 4-like, L-type lectin-domain-containing receptor kinase, G-type lectin S-receptor-like serine, B. rapa photosystem II 22 kDa protein, and MLP-like protein, showed significant upregulation. In the secondary infection stage, 45 of 50 overlapping DEGs were upregulated. These upregulated DEGs included the predicted B. rapa endoglucanase, long-chain acyl-CoA synthetase, WRKY transcription factor, NAC domain-containing protein, cell division control protein, auxin-induced protein, and protein variation in compound-triggered root growth response-like and xyloglucan glycosyltransferases. In both the primary and secondary infection stages, the DEGs were predicted to be Brassica rapa putative disease resistance proteins, L-type lectin domain-containing receptor kinases, ferredoxin-NADP reductases, 1-aminocyclopropane-1-carboxylate synthases, histone deacetylases, UDP-glycosyltransferases, putative glycerol-3-phosphate transporters, and chlorophyll a-binding proteins, which are closely associated with plant defense responses, biosynthetic processes, carbohydrate transport, and photosynthesis. This study revealed the pivotal role of transcription factors in the initiation of infection and establishment of intracellular parasitic relationships during the primary infection stage, as well as the proliferation and differentiation of the pathogen within the host cell during the secondary infection stage.

The role of the Rho family small GTPases in regulation of normal and pathological processes

Small GTPases are small (about 21 kDa) proteins that regulate many biological processes, such as vesicle transport, cell division cycle, cell migration, invasion, adhesion, proliferation and DNA repair, they are involved in carcinogenesis and neurodegenerative diseases. Some of these proteins, like those in the Rho family, are important regulators of the actin cytoskeleton, which has an impact on cell adhesion and motility. The review considers normal and pathological processes in human cells, which are regulated by the Rho family small GTPases. Particular attention is paid to inhibitors of small GTPases and their use in the treatment of various diseases.

Study use of Interferon Gamma-Induced Protein 10 Kda and C-Reactive Protein as Diagnostic Markers for Spontaneous Bacterial Peritonitis

Study use of Interferon Gamma-Induced Protein 10 Kda and C-Reactive Protein as Diagnostic Markers for Spontaneous Bacterial Peritonitis

Targeted Antisense Oligonucleotide-Mediated Skipping of Murine Postn Exon 17 Partially Addresses Fibrosis in D2.mdx Mice.

Periostin, a multifunctional 90 kDa protein, plays a pivotal role in the pathogenesis of fibrosis across various tissues, including skeletal muscle. It operates within the transforming growth factor beta 1 (Tgf-β1) signalling pathway and is upregulated in fibrotic tissue. Alternative splicing of Periostin's C-terminal region leads to six protein-coding isoforms. This study aimed to elucidate the contribution of the isoforms containing the amino acids encoded by exon 17 (e17+ Periostin) to skeletal muscle fibrosis and investigate the therapeutic potential of manipulating exon 17 splicing. We identified distinct structural differences between e17+ Periostin isoforms, affecting their interaction with key fibrotic proteins, including Tgf-β1 and integrin alpha V. In vitro mouse fibroblast experimentation confirmed the TGF-β1-induced upregulation of e17+ Periostin mRNA, mitigated by an antisense approach that induces the skipping of exon 17 of the Postn gene. Subsequent in vivo studies in the D2.mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that our antisense treatment effectively reduced e17+ Periostin mRNA expression, which coincided with reduced full-length Periostin protein expression and collagen accumulation. The grip strength of the treated mice was rescued to the wild-type level. These results suggest a pivotal role of e17+ Periostin isoforms in the fibrotic pathology of skeletal muscle and highlight the potential of targeted exon skipping strategies as a promising therapeutic approach for mitigating fibrosis-associated complications.

Structural and functional characterization of Hdh-HSBP1 and its involvement in heat stress and early development in Pacific abalone, Haliotis discus hannai

Heat shock factor binding protein 1 (HSBP1) is known to regulate the activity of heat shock factor 1 (HSF1) and the early development of organisms. To understand the involvement of HSBP1 in the heat shock response and embryonic and larval development of Pacific abalone (Haliotis discus hannai), the Hdh-HSBP1 gene was sequenced from the digestive gland (DG) tissue. The full-length sequence of Hdh-HSBP1 encompassed 738 nucleotides, encoding an 8.42 kDa protein consisting of 75 deduced amino acids. The protein contains an HSBP1 domain and a coiled-coil domain, which are conserved features in the HSBP1 protein family. Protein-protein molecular docking revealed that the coiled-coil region of Hdh-HSBP1 binds to the coiled-coil region of Hdh-HSF1. Tissue expression analysis demonstrated that the highest Hdh-HSBP1 expression occurred in the DG, whereas seasonal expression analysis revealed that this gene was most highly expressed in summer. In heat-stressed abalone, the highest expression of Hdh-HSBP1 occurred at 30 °C. Moreover, time-series analysis revealed that the expression of this gene began to increase significantly at 6 h post-heat stress, with higher expression observed at 12 h and 24 h post-heat stress. Furthermore, Hdh-HSBP1 mRNA expression showed a link to ROS production. Additionally, the expression of Hdh-HSBP1 showed significantly higher expression in the early stages of embryonic development in Pacific abalone. These results suggest that Hdh-HSBP1 plays a crucial role in the stress physiology of Pacific abalone by interacting with Hdh-HSF1, as well as its embryonic development.

Tick-borne pathogens isolated from ticks, rodents, and a shrew in Gangwon and Gyeonggi provinces in the Republic of Korea.

The prevalence of tick-borne pathogens (TBP), Orientia tsutsugamushi, Rickettsia and Borrelia spp. in wild small animals, namely wild rodents, is now widely investigated. This study is to present the prevalence and distribution of O. tsutsugamushi, Rickettsia and Borrelia spp. in wild small animals and ticks collected from Gyeonggi and Gangwon provinces, Republic of Korea (ROK) in 2014. A total of 131 wild small animals, rodents and shrews, and 2,954 ticks were collected from Gyeonggi and Gangwon provinces from May to November 2014. The wild small animals (KR1-9) and ticks (K1-17) were grouped in accordance with capture dates and locations. Among the wild small animals, a total of 393 tissues and blood samples were extracted from six selected small animal series (KR1-3, KR6-8). Also, each date and location-grouped ticks were identified for its species and pooled according to the stage of development. Molecular identification for Rickettsia, Orientia, and Borrelia species was performed using polymerase chain reaction (PCR). To detect TBPs among wild small animals and ticks, primer sets targeting the 56 kDa protein encoding gene of Orientia spp., outer membrane protein B gene (OmpB) of Rickettsia spp., and 5S-23S intergenic spacer region (IGS) gene of Borrelia spp. were used. Of the 393 wild small animals' blood and tissue samples, 199 (50.6%) were positive for Orientia spp., 158 (40.2%) were positive for Borrelia spp., and 55 (14.0%) were positive for Rickettsia spp. Moreover, a total of 14 tick pools (n = 377) was positive for Rickettsia spp. (n=128, 34.0%) and Borrelia spp. (n=33, 8.8%). High prevalence of Orientia spp. and Rickettsia spp. in rodents and shrews were observed. This study presents significant insights by presenting data collected in 2014 that the prevalence of TBP was already high in mid 2010s. This study highlights the sustainable routine surveillance model for TBP.

DNA methylase 1 influences temperature responses and development in the invasive pest Tuta absoluta.

DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absolutaMeyrick (Lepidoptera: Gelechiidae)-a destructive invasive pest-can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real-time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full-length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node-conserved structural features and six conserved DNA-binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double-stranded RNA and exposing Tuta absoluta to 35°C heat shock or -12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1-mediated temperature tolerance. This study advances the research on 'thermos Dnmt' and provides a potential target for RNAi-driven regulation of Tuta absoluta.

Identification and analysis of immunoreactive proteins of Shigella flexneri in human sera and stool specimens.

The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.

Gene Editing for Unraveling the Regulatory Role of a HER2-Associated Enhancer with lncRNA GAS5 and Related Genes in Breast Cancer Cells

Background: The HER2/neu gene belongs to the epidermal growth factor receptor (EGFR) family and encodes a 185 kDa protein with tyrosine kinase activity. HER2 amplification or overexpression has been associated with tumorigenesis and cancer progression, notably in breast cancer (BC). The long non-coding RNA GAS5 has been recognized as a tumor suppressor in BC, promoting apoptosis and inhibiting cell proliferation. Moreover, GAS5 regulates key signaling pathways in breast cancer, including PI3K/AKT/mTOR and PTEN signaling. The PI3K/AKT pathway is critical in various cancer processes, such as proliferation, apoptosis, invasion, and drug resistance. Breast cancer stem cells (BCSCs) and stemness genes also contribute to tumor growth and therapy resistance. Therefore, targeting key regulatory regions in the HER2 gene could offer potential therapeutic strategies. Objectives: In this study, we aimed to investigate the regulatory role of a putative enhancer located in the HER2 gene in the 17q12 region (GH17J039694; we referred to it as Her2-Enhancer1 in our previous study) on HER2 variants and genes that are connected to it through different pathways. Methods: We analyzed the effects of enhancer knockout on HER2 variants, GAS5, PTEN, and stemness genes in HER2-positive and HER2-negative breast cancer cells using the CRISPR/Cas9 system and real-time PCR. Results: Our results showed significantly reduced HER2 variant expression in both cell lines after genetic editing. Furthermore, the expression of some other target genes was affected, including GAS5, P53, and PTEN. Interestingly, we observed altered expression of stemness genes like NANOG, SOX2, and OCT4, indicating possible implications for cancer stem cell properties. Conclusions: Our study highlights the potential regulatory role of the studied region as an enhancer in the HER2 gene and sheds light on its influence on various cancer-related pathways, providing valuable insights into breast cancer pathogenesis and potential therapeutic targets.

Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight.

A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H-13C correlated NMR spectra of a 50kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.

Proteomic profiling of peanut hairy root culture unveils distinctive adaptive responses induced by elicitor treatment across diverse time intervals

Plants employ a diverse array of responses to counteract and defend against stressors. Utilizing quantitative proteomic analysis allows for the exploration of global proteomic dynamics in cells and tissues at specific time points. In order to comprehend the intricate mechanisms underlying plant responses, we conducted a proteomic investigation of peanut hairy root cultures induced with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD) at multiple time points. The study identified 292, 317, and 327 differentially expressed proteins (DEPs) in hairy root tissues at 24-, 48-, and 72 h intervals post-elicitation, respectively. Principal component analysis (PCA) distinctly clustered each timepoint of treatment and the control group. Gene ontology (GO) enrichment analysis highlighted terms such as "responding to biotic stimuli" and "defense response" during the early response, with the upregulation of Heat shock cognate 70 kDa protein, Wound-induced protein 1, and Disease resistance-like protein. The 72 h time point specifically showed an upregulation of superoxide dismutase (SOD) and auxin response factor, indicating a delayed response and illustrating how hairy roots adapt to stress in later stages. Furthermore, GO enrichment at later stages implicated alterations in protein production, involving both synthesis and degradation processes. Proteolytic enzymes, including those in the ubiquitin/proteasome pathway, were identified as facilitating protein degradation. The detection of small molecular weight (MW) peptides in the culture medium of elicited hairy roots suggested their origin from the proteolytic cleavage of precursor proteins. The GO enrichment of culture medium peptides pointed towards involvement in signaling pathways. The suggested signaling cascade acts as a potential link that connects the recognition of stress to the initiation of appropriate response to external stressors. This approach delves into dynamic changes in protein expression and secretion patterns over various intervals, providing insights into the plant's adaptive response to external stress.

The unique properties of Big tau in the visual system.

Tau is a microtubule associated protein that plays important roles in regulating the properties of microtubules and axonal transport, as well as tauopathies associated with toxic aggregates leading to neurodegenerative diseases. It is encoded by the MAPT gene forming multiple isoforms (45-60 kDa) by alternative splicing which are developmentally regulated. The high molecular weight (MW) tau isoform of 105 kDa, termed Big tau, was originally discovered in the peripheral nervous system (PNS) but later found in selective CNS areas. It contains an additional large exon 4a generating a long projecting domain of about 250 amino acids. Here we investigated the properties of Big tau in the visual system of rats, its distribution in retinal ganglion cells and the optic nerve as well as its developmental regulation using biochemical, molecular and histological analyses. We discovered that Big tau is expresses as a 95 kDa protein (termed middle MW) containing exons 4a, 6 as well as exon 10 which defines a 4 microtubule-binding repeats (4R). It lacks exons 2/3 but shares the extensive phosphorylation characteristic of other tau isoforms. Importantly, early in development the visual system expresses only the low MW isoform (3R) switching to both the low and middle MW isoforms (4R) in adult retinal ganglion neurons and their corresponding axons. This is a unique structure and expression pattern of Big tau, which we hypothesize is associated with the specific properties of the visual system different from what has been previously described in the PNS and other areas of the nervous system.

Amsacta moorei entomopoxvirus encodes a protein kinase with dual activity and a broad substrate spectrum including two putative cellular substrates.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

Gene cloning, expression, and enzyme kinetics analysis of Eimeria tenella 2- methylcitrate synthase

In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the 2-MCC pathway of Eimeria tenella. The analysis of genomic data found that the coding gene of 2- methylcitrate synthase (EC 2.3.3.5, PrpC) exists in E. tenella, which is a key enzyme of 2-MCC pathway. Through the search analysis of the database (ToxoDB), it was found that ETH_ 00026655 contains the complete putative sequence of EtprpC. In this study, we amplified the ORF sequence of EtprpC based on putative sequence. Then, prokaryotic expression, enzyme activity and kinetic analysis was performed. The results showed that the EtprpC ORF sequence was 1272 bp, encoding a 46.3 kDa protein comprising 424 amino acids. Enzyme activity assays demonstrate linearity between the initial reaction rate (OD/min) and EtPrpC concentration (ranging from 1.5 to 9 µg/reaction), with optimal enzyme activity observed at 41°C and pH 8.0. The results of enzymatic kinetic analysis showed that the Km of EtPrpC for propionyl-CoA, oxaloacetic acid, and acetyl-CoA was 5.239 ± 0.17 mM, 1.102 ± 0.08 μM, and 5.999 ± 1.24 μM, respectively. The Vmax was 191.11 ± 19.1 nmol/min/mg, 225.48 ± 14.4 nmol/min/mg, and 370.02 ± 25.8 nmol/min/mg when EtPrpC concentration at 4, 6, and 8 μg, respectively. Although the ability of EtPrpC to catalyze acetyl-CoA is only 0.11% of its ability to catalyze propionyl-CoA, it indicates that the 2-MCC pathway in E. tenella is similar to that in bacteria and may have a bypass function in the TCA cycle. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs.

Effects of air frying on protein digestive properties in scallop (Patinopecten yessoensis) adductor muscles and the mechanisms involved

The effects of air frying at different temperatures (140 °C, 160 °C, 180 °C, and 200 °C) on the simulated digestive properties of proteins in scallop adductor muscle (SAM) and the mechanisms involved were investigated. The results of the simulated gastrointestinal digestion showed that the 160 °C-treated SAM group had significantly higher protein digestibility than the fresh SAM group, which was reflected in higher degree of hydrolysis, more low molecular weight (<0.5 kDa) protein digestive products, and less insoluble residues, while the 140 °C-, 180 °C- and 200 °C-treated SAM groups all had opposite results. Moreover, air frying altered the solubility, secondary structure percentage and surface hydrophobicity of SAM proteins, as well as the microstructure of insoluble residues in simulated gastric juice. Correlation analysis showed that the protein digestive level of the air fried SAMs was positively correlated with the ratio of soluble proteins before digestion and the surface hydrophobicity of insoluble residues, but negatively correlated with the content of α-helix of soluble proteins dissolved in simulated gastric juice. Therefore, the highest digestibility of the 160 °C-treated SAM group can be attributed to the lowest α-helix content of soluble proteins, as well as the highest surface hydrophobicity and the largest pores size of insoluble residues. The present study provides a good basis for understanding the effects of air frying temperature on the digestive properties of aquatic muscle foods and the mechanisms involved, which contributes to establish precise air frying method for aquatic muscle foods.

X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo' proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.