The protein journal最新文献

Tissue plasminogen activator - plasminogen activator inhibitor complex (tPAIc) is a critical biomarker to assess fibrinolytic dysfunction, which is widely used in clinics. Quality control material (QCM) plays an important role in immunoassays for human tPAIc. The QCM of tPAIc are derived from human plasma with many disadvantages. Recombinant protein is a promising substitute for human plasma to work as the source of QCM. However, tPAIc is a protein complex, consisting of three parts, tPA-A, tPA-B, and PAI-1, which makes the expression more difficult. This study aimed to obtain recombinant tPAIc QCM with excellent performance for immunoassay. Three recombinant plasmids that matched each part of the protein complex were constructed and co-transfected to HEK293F cells. The optimal molar ratio of three plasmids was further explored. Each part of the proteins was secreted from cells and the target protein tPAIc was self-assembled in the supernatant. After being identified by western blot and chemiluminescent immunoassay (CLIA), calculating the concentration of tPAIc in the supernatant, tPAIc was diluted to approximately 50 ng/mL and 5 ng/mL, distributed, and lyophilized in ampoules, working as QCM in tPAIc immunoassay. The homogeneity, stability, and recovery of the QCM were further evaluated. The three plasmids were successfully constructed. The target protein complex, tPAIc, was obtained in the supernatant at about 6500 ng/mL, under the best three plasmids co-transfected molar ratio 1:1:1. The QCMs were uniform in different ampoules. They were verified to be highly stable for at least 1 year when stored at 4 ℃ and - 20 ℃. The recombinant tPAIc QCMs for immunoassay were obtained with high quality to replace plasma-derived QCMs, which provides valuable insight into more application scenarios of recombinant proteins.

The transactive response DNA binding protein 43 (TDP-43) is an RNA/DNA-binding protein that is involved in a number of cellular functions, including RNA processing and alternative splicing, RNA transport and translation, and stress granule assembly. It has attracted significant attention for being the primary component of cytoplasmic inclusions in patients with amyotrophic lateral sclerosis or frontotemporal dementia. Mounting evidence suggests that both cytoplasmic aggregation of TDP-43 and loss of nuclear TDP-43 function contribute to TDP-43 pathology. Furthermore, recent studies have demonstrated that TDP-43 is an important component of many constitutive or stress-induced biomolecular condensates. Dysregulation or liquid-to-gel transition of TDP-43 condensates can lead to alterations in TDP-43 function and the formation of TDP-43 amyloid fibrils. In this review, we summarize recent research progress on the structural characterization of TDP-43 and the TDP-43 phase transition. In particular, the roles that disease-associated genetic mutations, post-translational modifications, and extrinsic stressors play in the transitions among TDP-43 monomers, liquid condensates, solid condensates, and fibrils are discussed. Finally, we discuss the effectiveness of available regulators of TDP-43 phase separation and aggregation. Understanding the underlying mechanisms that drive the pathological transformation of TDP-43 could help develop therapeutic strategies for TDP-43 pathology.

Butyrylcholinesterase (BChE; EC 3.1.1.8), a serine hydrolase found in various tissues, hydrolyses choline esters such as acetylcholine and succinylcholine, as well as other esters such as heroin and acetylsalicylic acid. It is considered to play a role in lipid metabolism as it belongs to the same enzyme group as lipases and its catalytic subunits are similar. In this study, the effects of unsaturated fatty acids, namely arachidonic (AA), linoleic (LA), alpha-linolenic (ALA) and oleic acid (OA), on equine serum BChE (EqBChE) were investigated. Enzyme activity was measured by the modified Ellman method. When the activity results were evaluated, the IC50 values were found 45.49, 8.465, 1556, and 56.57 μM; while the Ki values were 63.92, 11.46, 1800, and 15.24 μM for AA, ALA, LA, and OA, respectively. Analysis of the kinetic results showed that ALA was compatible with mixed inhibition and other fatty acids were compatible with non-competitive inhibition, a special type of mixed inhibition. Molecular docking predicted binding of the fatty acids to the active site, as well as to predicted allosteric sites. The results of this study provide another support to the hypothesis that cholinesterases are associated with lipid metabolism.

Factors that impede the normal growth and development of plants are termed 'stress factors' and result in yield loss. Exposure to elevated concentrations of heavy metals has been demonstrated to induce the production of reactive oxygen species (ROS), which can affect physiological and biochemical processes. The present study investigated the effects of two common heavy metals (Hg²⁺ and Cd²⁺) on specific physiological and biochemical parameters of sorghum (Sorghum bicolor (L.) Moench). The study utilized doses ranging from 3 to 15 ppm for the application of heavy metals. The study focused on the effects of these metals on the activities of enzymes (superoxide dismutase and peroxidase), chlorophyll content, fresh weight, dry weight, and proline content in sorghum plants. The study found that applying mercury at 9 ppm resulted in the highest activity of peroxidase (POX), with an observed increase of 69.57% compared to the control. Conversely, cadmium application at 12 ppm elicited the highest activity, increasing by 102.17% compared to the control. For superoxide dismutase (SOD), the peak activity was observed at 6 ppm for both applications, with an increase of 84.16% in the mercury application control group and 121.08% in the cadmium application group compared to the control group. A similar pattern was observed in the chlorophyll content, which initially increased and then decreased. Declines in fresh weight, dry weight, and proline accumulation were also documented.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder and its concurrent presence with chronic kidney disease (CKD) is a significant concern. Glycerol kinase (GK) and glycerol 3-phosphate shuttle enzymes (cGPDH and mGPDH) facilitate the regulation of endogenous glucose production in many cell lines. This research investigates the functions of GK, cGPDH, and mGPDH in HEK293 cells. Standard protocols were employed to assess enzyme activity, mRNA- and protein-expression, glucose uptake, and production. Homology modeling and molecular docking were employed to elucidate interactions of genistein and metformin with these enzymes. The secondary structures of GK, cGPDH and mGPDH and the thermal stability of cGPDH and mGPDH were analyzed by CD spectra. Genistein inhibited GK activity by 40%, while metformin decreased cGPDH and mGPDH activity by 58% and 55%, respectively, in HEK293 cells. Nonetheless, the expression levels of mRNA and protein remained unaltered. Genistein and metformin inhibited HEK293 glucose production by 0.46-fold and 0.63-fold, respectively. Genistein reduced glucose uptake by 0.26-fold, while metformin increased it by 0.51-fold. Genistein allosterically interacted with GK with a CDocker energy of -27.71, while metformin interacted with Gln295 and Lys296 of the catalytic loop of cGPDH and the FAD⁺ binding domain of mGPDH, yielding CDocker energies of -11.12 and -13.34, respectively. This study indicated the role of genistein and metformin on GK, cGPDH, and mGPDH in HEK293 cells.

Malaria remains a formidable challenge to global health, claiming the lives of nearly half a million individuals annually despite vigorous efforts to curb its spread. Among the myriad factors influencing the persistence and virulence of this disease, the role of specific proteins during the Plasmodium development cycle is critical. The protein of interest, Pfs16, is a Parasitophorous Vacuole Membrane Protein expressed from the earliest asexual stages, which encompass the development of Plasmodium falciparum in the host, to the final stage of the parasite's development in the mosquito, the sporozoite, playing a crucial role in this lifecycle. Understanding the function and mechanism of this conserved protein is pivotal for advancing our strategies to combat malaria. In this review, we examine the work on Pfs16 in both the asexual and sexual stages of parasite development, aiming to gain a better understanding of this protein as a promising candidate for drug and vaccine development.

Plant-derived cysteine proteases have emerged as a compelling subject of investigation, capturing scientific interest owing to their potential applications in diverse industries, including food and biotechnology. This study focused on isolating Kaempferia galanga cysteine protease (KgCP) from rhizomes of Kaempferia galanga, followed by a comprehensive characterization of the protease. It was purified and characterized using various biochemical and biophysical techniques, including anion-exchange chromatography, gel filtration, SDS-PAGE electrophoresis, and enzyme assays. With a yield of 23.2%, the purification process generated a 6.03-fold increase in specific activity. KgCP's molecular weight was determined to be around 33 kDa and exhibited optimal catalytic performance at 55 °C and pH 5.5. Values of its catalytic parameters, V_max and K_m, were found to be 103.7 Units min^-1 and 0.025 μmol, respectively. Inhibition of KgCP by various cysteine protease inhibitors - E-64, iodoacetamide, and mercury chloride confirmed it to be a cysteine protease. The inclusion of detergents and organic solvents did not affect the stability of KgCP. Although proteolytic activity was compromised by metal ions such as Cd²⁺, Co²⁺, and Fe³⁺, other metal ions (Ca²⁺, Mg²⁺, Mn²⁺, Sn²⁺, Sr²⁺, etc.) showed negligible impact on its proteolytic activity. These findings expand our understanding of the biological characteristics of this cysteine protease, highlighting its potential for applications in the dairy industry, bioactive peptide synthesis, detergents industry, etc. The entire work can be graphically presented as follows.

Thioredoxin-like ferredoxin is a small homodimeric protein containing a [2Fe-2S] cluster in each monomer. It is only found in bacteria but its physiological function remains largely unknown. The cobalamin biosynthetic operon in the genome of the purple phototroph Rhodobacter capsulatus encodes a putative ferredoxin dubbed as CfrX. To characterize this protein, we cloned, expressed, purified, and crystalized the recombinant CfrX in the iron-sulfur cluster-bound state, and solved the structure at 2.1-Å resolution. Adopting a typical thioredoxin-like ferredoxin fold, a CfrX monomer binds one [2Fe-2S] cluster through four Cys residues located on two protruding loops. Unexpectedly, CfrX dimerizes in a previously unreported manner. With the structural information, we ascertained CfrX as a thioredoxin-like ferredoxin. While the precise function of CfrX in cobalamin biosynthesis is elusive, a link between CfrX and aerobic cobaltochelatase should exist due to the gene clustering pattern. We also discussed the possible relationship among CfrX, CobW, and CobNST with respect to the [2Fe-2S] cluster.

In this study, we identified and partially purified antimicrobial peptides belonging to the family of lipid transfer proteins (LTPs) from Capsicum chinense seeds (UENF 1751 accession). Fractions rich in LTPs were obtained via ion exchange chromatography and subsequently purified via reverse-phase chromatography in an HPLC system. Therefore, two fractions were revealed: C1 (the nonretained fraction) and C2 (the retained fraction in ion-exchange chromatography). Fraction C1 was subjected to reverse-phase chromatography via a C18 column on an HPLC system, and ten fractions were obtained (P1-P10), all of which significantly inhibited the growth of Candida albicans, except for P4 and P9. The viability analysis of the active fractions at a concentration of 100 µg.mL^-1 against C. albicans revealed that they did not exhibit fungicidal activity but rather exhibited fungistatic activity. The peptide is considered fungicidal when it results in the total loss of viable yeast cells, that is, when it causes the complete death of the fungi. When the substance only inhibits cell growth, but does not eliminate them completely, the effect is classified as fungistatic. Fractions P3, P4, P7, and P10 inhibited Tenebrio molitor larvae α-amylase. The P10 fraction presented protein bands in its electrophoretic profile with a molecular mass between 6.5 kDa and 14.2 kDa and reacted positively to an antibody produced against a protein from the LTP family bywestern blotting. The results of the analysis of amino acid residues from the P10 fraction revealed similarity between type I LTPs and type II LTPs. The ultrastructural aspects of C. albicans cells exposed to the P10 fraction were evaluated via transmission electron microscopy (TEM), with significant differences in their morphology being evident compared with those of the control. In summary, our results demonstrated the presence of LTPs in C. chinense seeds with inhibitory effects on the growth of yeasts of the genus Candida, which exhibited fungistatic effects and structural changes in C. albicans cells, in addition to exhibiting inhibitory effects on the larval insect T. molitor α-amylase.