The protein journal最新文献

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbβ3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher K_m values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).

Protein-DNA and protein-RNA interactions are involved in many biological processes and regulate many cellular functions. Moreover, they are related to many human diseases. To understand the molecular mechanism of protein-DNA binding and protein-RNA binding, it is important to identify which residues in the protein sequence bind to DNA and RNA. At present, there are few methods for specifically identifying the binding sites of disease-related protein-DNA and protein-RNA. In this study, so we combined four machine learning algorithms into an ensemble classifier (EPDRNA) to predict DNA and RNA binding sites in disease-related proteins. The dataset used in model was collated from UniProt and PDB database, and PSSM, physicochemical properties and amino acid type were used as features. The EPDRNA adopted soft voting and achieved the best AUC value of 0.73 at the DNA binding sites, and the best AUC value of 0.71 at the RNA binding sites in 10-fold cross validation in the training sets. In order to further verify the performance of the model, we assessed EPDRNA for the prediction of DNA-binding sites and the prediction of RNA-binding sites on the independent test dataset. The EPDRNA achieved 85% recall rate and 25% precision on the protein-DNA interaction independent test set, and achieved 82% recall rate and 27% precision on the protein-RNA interaction independent test set. The online EPDRNA webserver is freely available at http://www.s-bioinformatics.cn/epdrna .

Protein-protein interactions (PPIs) involve the physical or functional contact between two or more proteins. Generally, proteins that can interact with each other always have special relationships. Some previous studies have reported that gene ontology (GO) terms are related to the determination of PPIs, suggesting the special patterns on the GO terms of proteins in PPIs. In this study, we explored the special GO term patterns on human PPIs, trying to uncover the underlying functional mechanism of PPIs. The experimental validated human PPIs were retrieved from STRING database, which were termed as positive samples. Additionally, we randomly paired proteins occurring in positive samples, yielding lots of negative samples. A simple calculation was conducted to count the number of positive samples for each GO term pair, where proteins in samples were annotated by GO terms in the pair individually. The similar number for negative samples was also counted and further adjusted due to the great gap between the numbers of positive and negative samples. The difference of the above two numbers and the relative ratio compared with the number on positive samples were calculated. This ratio provided a precise evaluation of the occurrence of GO term pairs for positive samples and negative samples, indicating the latent GO term patterns for PPIs. Our analysis unveiled several nuclear biological processes, including gene transcription, cell proliferation, and nutrient metabolism, as key biological functions. Interactions between major proliferative or metabolic GO terms consistently correspond with significantly reported PPIs in recent literature.

As the demand for immunotherapy to treat and manage cancers, infectious diseases and other disorders grows, a comprehensive understanding of amino acids and their intricate role in antibody engineering has become a prime requirement. Naturally produced antibodies may not have the most suitable amino acids at the complementarity determining regions (CDR) and framework regions, for therapeutic purposes. Therefore, to enhance the binding affinity and therapeutic properties of an antibody, the specific impact of certain amino acids on the antibody's architecture must be thoroughly studied. In antibody engineering, it is crucial to identify the key amino acid residues that significantly contribute to improving antibody properties. Therapeutic antibodies with higher binding affinity and improved functionality can be achieved through modifications or substitutions with highly suitable amino acid residues. Here, we have indicated the frequency of amino acids and their association with the binding free energy in CDRs. The review also analyzes the experimental outcome of two studies that reveal the frequency of amino acids in CDRs and provides their significant correlation between the outcomes. Additionally, it discusses the various bond interactions within the antibody structure and antigen binding. A detailed understanding of these amino acid properties should assist in the analysis of antibody sequences and structures needed for designing and enhancing the overall performance of therapeutic antibodies.

Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.

Cecropin A (1-7) is a cationic antimicrobial peptide which contain lots of basic amino acids. To understand the effect of basic amino acids on cecropin A (1-7), analogues CA2, CA3 and CA4 which have more arginine or lysine at the N-terminal or C-terminal were designed and synthesized. The interaction of cecropin A (1-7) and its analogs with DNA was studied using ultraviolet-visible spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Multispectral analysis showed that basic amino acids improved the interaction between the analogues and DNA. The interaction between CA4 and DNA is most pronounced. Fluorescence spectrum indicated that Ksv value of CA4 is 1.19 × 10⁵  L mol^-1 compared to original peptide cecropin A (1-7) of 3.73 × 10⁴  L mol^-1. The results of antimicrobial experiments with cecropin A (1-7) and its analogues showed that basic amino acids enhanced the antimicrobial effect of the analogues. The antimicrobial activity of CA4 against E. coli was eightfold higher than that of cecropin A (1-7). The importance of basic amino acid in peptides is revealed and provides useful information for subsequent studies of antimicrobial peptides.

The hydrolysis of deacylated glycerophospholipids into sn-glycerol 3-phosphate and alcohol is facilitated by evolutionarily conserved proteins known as glycerophosphodiester phosphodiesterases (GDPDs). These proteins are crucial for the pathogenicity of bacteria and for bioremediation processes aimed at degrading organophosphorus esters that pose a hazard to both humans and the environment. Additionally, GDPDs are enzymes that respond to multiple nutrients and could potentially serve as candidate genes for addressing deficiencies in zinc, iron, potassium, and especially phosphate in important plants like rice. In mammals, glycerophosphodiesterases (GDEs) play a role in regulating osmolytes, facilitating the biosynthesis of anandamine, contributing to the development of skeletal muscle, promoting the differentiation of neurons and osteoblasts, and influencing pathological states. Due to their capacity to enhance a plant's ability to tolerate various nutrient deficiencies and their potential as pharmaceutical targets in humans, GDPDs have received increased attention in recent times. This review provides an overview of the functions of GDPD families as vital and resilient enzymes that regulate various pathways in bacteria, plants, and humans.

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.