-
International Journal of Biochemistry... 2024Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective...
INTRODUCTION
Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP.
METHODS
Codon-optimized human CD2AP was expressed in Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP.
RESULTS
Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains.
CONCLUSION
We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.
PubMed: 38765876
DOI: 10.62347/UVSH8436 -
Food Chemistry: X Jun 2024Knowledge regarding the denaturation process and control methods for depolymerized sol-state myofibrillar proteins (MPs) during freezing remains scant. This study...
Effects of different protein cross-linking degrees on physicochemical and subsequent thermal gelling properties of silver carp myofibrillar proteins sol subjected to freeze-thaw cycles.
Knowledge regarding the denaturation process and control methods for depolymerized sol-state myofibrillar proteins (MPs) during freezing remains scant. This study investigated the effects of protein cross-linking treatment before freezing on physicochemical and subsequent gelation properties of MPs sol subjected to freeze-thaw (F-T) cycles. Results indicated that after five F-T cycles, cross-linked MPs sols showed increased high molecular weight polymers and bound water (T and T) mobility, suggesting enhanced protein-protein interactions at the expense of protein-water interactions. Upon heating after F-T cycles, gels formed from cross-linked sols exhibited significantly higher hardness, springiness, and cooking loss ( < 0.05), alongside more contracted gel networks. Correlation analysis revealed that the formation and properties of thermal gel after freezing closely relate to changes in molecular conformation and chemical bonds of cross-linked MPs sol during freezing. This study provides new insights into regulating the freezing stability and post-thawed thermal processing properties of sol-based surimi products.
PubMed: 38764785
DOI: 10.1016/j.fochx.2024.101448 -
ACS Omega May 2024Protein-scaffolded atomically precise metal nanoclusters (NCs) have emerged as a promising class of biofriendly nanoprobes at the forefront of modern research,...
Protein-scaffolded atomically precise metal nanoclusters (NCs) have emerged as a promising class of biofriendly nanoprobes at the forefront of modern research, particularly in the area of sensing. The photoluminescence (PL) intensity of several nanoclusters showed a systematic temperature-dependent fluctuation, but the mechanism remains ambiguous and is poorly understood. We tried to shed some light on this mechanistic aspect by testing a couple of hypotheses: (i) conformational fluctuation of the protein scaffold-mediated PL intensity fluctuation and (ii) PL intensity fluctuation due to the variation in the radiative and nonradiative transition rates. Herein, the PL intensity of the lysozyme-capped copper nanocluster (Lys-Cu NC) showed excellent temperature dependency; upon increasing the temperature, the PL intensity gradually decreased. However, contrasting effects can be seen when the nanocluster is exposed to a chemical denaturant (guanidine hydrochloride (GdnHCl)); the PL intensity increased with the increase in the GdnHCl concentration due to the change in the ionic strength of the medium. This discrepancy clearly suggests that the thermal PL intensity fluctuation cannot be explained by a change in the scaffold conformation. Furthermore, upon closer investigation, we observed a 2-fold increase in the nonradiative decay rate of the Lys-Cu NC at the elevated temperature, which could reasonably explain the decrease in the PL intensity of the nanocluster at the higher temperature. Additionally, from the result, it was evident that the protein scaffold-metal core interaction played a key role here in stabilizing each other; hence, the scaffold structure remained unaffected even in the presence of chemical denaturants.
PubMed: 38764622
DOI: 10.1021/acsomega.4c02223 -
Food Science of Animal Resources Mar 2024This study aimed to determine the effects of humectants on moisture content, water activity, tenderness, color, microbiological analysis, protein denaturation, and...
This study aimed to determine the effects of humectants on moisture content, water activity, tenderness, color, microbiological analysis, protein denaturation, and oxidation of jerky. A thorough search for papers published in scientific journals that examined the impacts of humectants on jerky was carried out using Web of Science, Google Scholar, PubMed, and Science Direct. Only 14 studies matched inclusion requirements. They were used in the meta-analysis to synthesise quantitative findings. In the current investigation, jerky produced with beef, poultry, goat, or pork was used. The standardised mean difference (SMD) between treatments with humectants and controls was examined to investigate the effects of humectants using random-effects models. Heterogeneity was investigated using meta-regression. A subgroup analysis was carried out for significant factors. Results revealed that the addition of humectants had no significant impact on water activity, pH, fat, ash, CIE L*, or CIE a* (p>0.05). However, humectant addition significantly increased moisture (SMD=1.28, p<0.05), CIE b* (SMD=1.67, p<0.05), and overall acceptability (SMD=1.73, p<0.05). It significantly decreased metmyoglobin (SMD=-0.96, p<0.05), shear force (SMD=-0.84, p<0.05), and protein (SMD=-1.61, p<0.05). However, it was difficult to get a firm conclusion about how humectants affected the myofibrillar fragmentation index, total plate count, and 2-thiobarbituric acid-reactive substances because there were fewer than ten studies. To sum up, the proper use of humectants in jerky demands careful attention to both type and quantity, needing a delicate balancing act with other contributing factors.
PubMed: 38764511
DOI: 10.5851/kosfa.2024.e3 -
Microbiology Spectrum Jun 2024Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is a traditional non-culture technique that can provide a fingerprint of the microbial...
UNLABELLED
Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is a traditional non-culture technique that can provide a fingerprint of the microbial community. In the field of gut microbiota analysis, PCR-DGGE still holds potential for development. In the present study, we utilized an improved nested PCR-DGGE approach targeting the V3 region of 16S ribosomal DNA to investigate the impact of whole grain highland hull-less barley (WHLB), a cereal known for its significant hypocholesterolemic effect, on the gut microbiota profiles of high-fat diet rats. Seventy-two male Sprague-Dawley rats were divided into four groups and fed a normal control diet, a high-fat diet, or a high-fat diet supplemented with a low or high dose of WHLB for 4 or 8 weeks. The results revealed that the dominant bands varied among different dose groups and further changed with different treatment times. The compositions of bacterial communities in feces and cecal content were similar, but the dominant bacterial bands differed. After performing double DGGE, extracting the bands, sequencing the DNA, and aligning the sequences, a total of 19 bands were classified under the Firmicutes and Bacteroidetes phyla, while two bands were identified as unclassified uncultured bacteria. The relative abundance of , Uncultured sp., and sp. increased following the administration of WHLB. Illumina-based sequencing was employed to assess the reliability of DGGE, demonstrating its reliability in analyzing the dominant taxonomic composition, although it may have limitations in accurately detecting the alpha diversity of bacterial species.
IMPORTANCE
While next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.
Topics: Animals; Hordeum; Male; Rats; Rats, Sprague-Dawley; Gastrointestinal Microbiome; Diet, High-Fat; Bacteria; Denaturing Gradient Gel Electrophoresis; RNA, Ribosomal, 16S; Whole Grains; Feces; DNA, Bacterial; Polymerase Chain Reaction; Cecum
PubMed: 38747621
DOI: 10.1128/spectrum.04089-23 -
Frontiers in Molecular Biosciences 2024The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the...
The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon it is capable of functioning over a wide range of temperatures. Existing X-ray structures were determined at temperatures where the protein is least active. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2°C-65°C) and metal ion binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbations with and without magnesium and manganese. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature. Hydrogen exchange protection factors and temperature coefficients reveal that both the thumb and finger domains are very dynamic relative to the palm and little-finger (LF) domains. These trends remain true at high temperature with dynamics increasing as temperatures increase from 35°C to 50°C. Notably, NMR spectra show that the Dbh tertiary structure cold denatures beginning at 25°C and increases in denaturation as the temperature is lowered to 5°C with little change observed by CD. Above 35°C, chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, without DNA bound. In contrast, these bound metals are not apparent in any Dbh crystal structures of the protein without DNA. Two ion binding sites are confirmed to be near the active site, as reported in other Y-family polymerases, and we report a novel ion binding site in the LF domain. Thus, the solution-state structure of the Dbh polymerase is distinct from that of the solid-state structures and shows an unusually high cold denaturation temperature.
PubMed: 38745908
DOI: 10.3389/fmolb.2024.1364068 -
Inorganic Chemistry May 2024Enzyme immobilization within metal-organic frameworks (MOFs) is a promising solution to avoid denaturation and thereby utilize the desirable properties of enzymes...
Enzyme immobilization within metal-organic frameworks (MOFs) is a promising solution to avoid denaturation and thereby utilize the desirable properties of enzymes outside of their native environments. The biomimetic mineralization strategy employs biomacromolecules as nucleation agents to promote the crystallization of MOFs in water at room temperature, thus overcoming pore size limitations presented by traditional postassembly encapsulation. Most biomimetic crystallization studies reported to date have employed zeolitic imidazole frameworks (ZIFs). Herein, we expand the library of MOFs suitable for biomimetic mineralization to include zinc(II) MOFs incorporating functionalized terephthalic acid linkers and study the catalytic performance of the enzyme@MOFs. Amine functionalization of terephthalic acids is shown to accelerate the formation of crystalline MOFs enabling new enzyme@MOFs to be synthesized. The structure and morphology of the enzyme@MOFs were characterized by PXRD, FTIR, and SEM-EDX, and the catalytic potential was evaluated. Increasing the linker length while retaining the amino moiety gave rise to a family of linkers; however, MOFs generated with the 2,2'-aminoterephthalic acid linker displayed the best catalytic performance. Our data also illustrate that the pH of the reaction mixture affects the crystal structure of the MOF and that this structural transformation impacts the catalytic performance of the enzyme@MOF.
Topics: Metal-Organic Frameworks; Temperature; Carboxylic Acids; Crystallization; Water; Phthalic Acids; Biomimetic Materials; Molecular Structure; Zinc; Enzymes, Immobilized; Amines; Catalysis
PubMed: 38743640
DOI: 10.1021/acs.inorgchem.4c00245 -
Heliyon May 2024Precise estimation of individual radiation dose utilizing biomaterials (fingernail, bone, and tooth) is very challenging due to their complex sample processing. Despite,...
Precise estimation of individual radiation dose utilizing biomaterials (fingernail, bone, and tooth) is very challenging due to their complex sample processing. Despite, tooth enamel, the most mineralized tissue of tooth is used for this purpose due to its high radiation sensitivity and ability to produce radiation induced long lived CO radicals. However, human teeth are not always available, and invasive nature of sample collection adds to the complexity making dose estimation difficult. In such cases, animal teeth (goat, cow, and moose) can be used as a substitute for human teeth due to comparable enamel sensitivity. Moreover, separation of enamel from dentine is a crucial step towards accurate dose estimation from irradiated teeth. In this work, Indian goat teeth were used as it was readily available to us and the comparison of goat enamel sensitivity to radiation was found to be within ∼7.4 % that of human. The enamel samples were separated following two chemical methods; (1) density separation using sodium polytungstate, (2) alkaline denaturation using NaOH and the quality was compared based on their purity and radiation sensitivity. Combined results of spectroscopic characterization using X-ray diffraction (XRD), Fourier transform infrared (FTIR), and Raman analysis authenticated the crystallinity and purity of the separated enamel samples. The radiation sensitivity of separated enamel samples was compared by electron paramagnetic resonance (EPR) analysis as a part of dosimetric characterization. The suitability of both the samples for retrospective dosimetry and epidemiological studies was checked by validating the dose estimated from separated enamel samples with standard alanine/EPR dosimeter.
PubMed: 38742072
DOI: 10.1016/j.heliyon.2024.e30571 -
Cureus Apr 2024Aim This study aims to synthesize silver nanoparticles (AgNPs) using herbal formulations derived from (ginger) and and to evaluate their anti-inflammatory and...
Green Synthesis of Silver Nanoparticles Using Zingiber officinale and Ocimum gratissimum Formulation for Its Anti-inflammatory and Antidiabetic Activity: An In Vitro Study.
Aim This study aims to synthesize silver nanoparticles (AgNPs) using herbal formulations derived from (ginger) and and to evaluate their anti-inflammatory and antidiabetic activities in vitro. Methods The synthesis of AgNPs was performed using and , and the AgNPs were confirmed by analyzing their ultraviolet-vis spectra. The anti-inflammatory activity was assessed using two assays, specifically the bovine serum albumin (BSA) denaturation assay and the egg albumin (EA) denaturation assay. The antidiabetic activity was assessed using the α-amylase inhibitory assay and the β-glucosidase inhibitory assay. Results This study evaluated the anti-inflammatory and antidiabetic activities of green-synthesized AgNPs using and . The maximum absorption peak was observed for AgNPs at ~433 nm (wavelength). In the BSA denaturation assay, 78% inhibition was observed at a concentration of 50 μl. Similarly, in the EA denaturation assay, an inhibition of 74% was observed at the same concentration compared to the standard. In terms of antidiabetic activity, when compared to the standard at a concentration of 50 μl, the α-amylase inhibitory assay and the β-glucosidase inhibitory assay showed approximately 78% and 80% inhibition, respectively. Conclusion The use of and extracts for the synthesis of AgNPs using a green synthesis method presents a sustainable and environmentally friendly approach. The synthesized AgNPs demonstrated significant anti-inflammatory and antidiabetic efficacy, suggesting their potential application in pharmaceuticals for treating diabetes and inflammation. Further research is necessary to investigate the effectiveness and safety of these substances in humans and to understand their underlying mechanisms of action.
PubMed: 38741855
DOI: 10.7759/cureus.58098 -
Journal of Biomedical Research May 2024The tumor suppressor p53 is a transcription factor with a powerful antitumor activity that is controlled by its negative regulator murine double minute 2 (MDM2, also...
The tumor suppressor p53 is a transcription factor with a powerful antitumor activity that is controlled by its negative regulator murine double minute 2 (MDM2, also termed HDM2 in humans) through a feedback mechanism. At the same time, is the most frequently mutated gene in human cancers. Mutant p53 proteins lose wild-type p53 tumor suppression functions but acquire new oncogenic properties, among which are deregulating cell proliferation, increasing chemoresistance, disrupting tissue architecture, and promoting migration, invasion and metastasis as well as several other pro-oncogenic activities. The oncogenic p53 mutation Y220C creates an extended surface crevice in the DNA-binding domain destabilizing p53 and causing its denaturation and aggregation. This cavity accommodates stabilizing small molecules that have therapeutic values. The development of suitable small-molecule stabilizers is one of the therapeutic strategies for reactivating the Y220C mutant protein. In this review, we summarize approaches that target p53-Y220C, including reactivating this mutation with small molecules that bind Y220C to the hydrophobic pocket and developing immunotherapies as the goal for the near future, which target tumor cells that express the p53-Y220C neoantigen.
PubMed: 38738269
DOI: 10.7555/JBR.37.20230093