Biochimica et biophysica acta. General subjects最新文献

Background: Immunotherapy is a powerful strategy for treating cancer and can be used to inhibit the post-surgical relapse of tumors. Methods: To achieve this, a Cell@hydrogel was developed as a template using a mixture of CT26 tumor cells and Pluronic® F-127/gelatin. Results: The proposed mixture has a solution-to-gelation functionality and vice versa. The morphology of the Cell@hydrogel was characterized by scanning electron microscopy and confocal microscopy. For photodynamic immunotherapy, the Cell@hydrogel was functionalized with Cy7 (Cy7-Cell@hydrogel) to quantify reactive oxygen species in CT26 tumor cells. Gel electrophoresis and membrane integrity tests were performed to determine the efficiency of the Cy7-Cell@hydrogel following photodynamic therapy. Conclusions: This protocol provides an alternative approach that mechanistically inhibits the post-surgical relapse of solid tumors based on immunotherapy.

Sonic hedgehog (Shh) is a morphogen with important roles in embryonic development and in the development of a number of cancers. Its activity is modulated by interactions with binding partners and co-receptors including heparin and heparin sulfate proteoglycans (HSPG). To identify antagonists of Shh/heparin binding, a diverse collection of 34,560 chemicals was screened in single point 384-well format. We identified and confirmed twenty six novel small molecule antagonists with diverse structures including four scaffolds that gave rise to multiple hits. Nineteen of the confirmed hits blocked binding of the N-terminal fragment of Shh (ShhN) to heparin with IC₅₀ values < 50 μM. In the Shh-responsive C3H10T1/2 cell model, four of the compounds demonstrated the ability to block ShhN-induced alkaline phosphatase activity. To demonstrate a direct and selective effect on ShhN ligand mediated activity, two of the compounds were able to block induction of Gli1 mRNA, a primary downstream marker for Shh signaling activity, in Shh-mediated but not Smoothened agonist (SAG)-mediated C3H10T1/2 cells. Direct binding of the two compounds to ShhN was confirmed by thermal shift assay and molecular docking simulations, with both compounds docking with the N-terminal heparin binding domain of Shh. Overall, our findings indicate that small molecule compounds that block ShhN binding to heparin and act to inhibit Shh mediated activity in vitro can be identified. We propose that the interaction between Shh and HSPGs provides a novel target for identifying small molecules that bind Shh, potentially leading to novel tool compounds to probe Shh ligand function.

Background

Resistant infectious diseases caused by gram-negative bacteria are among the most serious worldwide health problems. Antimicrobial peptides (AMPs) have been explored as promising antibacterial, antibiofilm, and anti-infective candidates to address these health challenges.

Major conclusions

Here we report the potent antibacterial effect of the peptide PaDBS1R6 on clinical bacterial isolates and identify an immunomodulatory peptide fragment incorporated within it. PaDBS1R6 was evaluated against Acinetobacter baumannii and Escherichia coli clinical isolates and had minimal inhibitory concentration (MIC) values from 8 to 32 μmol L⁻¹. It had a rapid bactericidal effect, with eradication showing within 3 min of incubation, depending on the bacterial strain tested. In addition, PaDBS1R6 inhibited biofilm formation for A. baumannii and E. coli and was non-toxic toward healthy mammalian cells. These findings are explained by the preference of PaDBS1R6 for anionic membranes over neutral membranes, as assessed by surface plasmon resonance assays and molecular dynamics simulations. Considering its potent antibacterial activity, PaDBS1R6 was used as a template for sliding-window fr agmentation studies (window size = 10 residues). Among the sliding-window fragments, PaDBS1R6F8, PaDBS1R6F9, and PaDBS1R6F10 were ineffective against any of the bacterial strains tested. Additional biological assays were conducted, including nitric oxide (NO) modulation and wound scratch assays, and the R6F8 peptide fragment was found to be active in modulating NO levels, as well as having strong wound healing properties.

General significance

This study proposes a new concept whereby peptides with different biological properties can be derived by the screening of fragments from within potent AMPs.

The pH varies in different tissues and organelles and also changes during some diseases. In this regard, the application of molecular switches that use a competition-based aptamer switch design in biological systems requires studying the thermodynamics of such systems at different pH values. In this work, we studied the binding of the classical ATP aptamer to ATP and competition strands under different pH and ionic conditions using fluorescent melting curve analysis. We have developed an original approach to processing source data from a PCR thermal cycler. It is based on constructing a thermodynamic model of the melting profile and the subsequent fit of experimental curves within this model. We have shown that this approach enables us to narrow the temperature region under study to the width of the melting region without a significant loss in the quality of the result. This impressively expands the application area of this approach compared to frequently used techniques that require mandatory measurement of the signal outside the melting region. The results obtained by the method showed that the thermodynamic parameters of the ATP aptamer and its duplexes with competition strands change depending on pH. Therefore, molecular switches that use a competition strand to the ATP aptamer may have a pH-dependent sensitivity that has not been previously considered. This should be taken into account for future rational design of similar systems.

Processing bodies (P-bodies, PBs) are cytoplasmic foci formed by condensation of translationally inactivated messenger ribonucleoprotein particles (mRNPs). Infection with the protozoan parasite Trypanosoma cruzi (T. cruzi) promotes PB accumulation in host cells, suggesting their involvement in host mRNA metabolism during parasite infection.

To identify PB-regulated mRNA targets during T. cruzi infection, we established a PB-defective human fibrosarcoma cell line by knocking out the enhancer of mRNA decapping 4 (EDC4), an essential component of PB assembly. Next-generation sequencing was used to establish transcriptome profiles for wild-type (WT) and EDC4 knockout (KO) cells infected with T. cruzi for 0, 3, and 24 h. Ingenuity pathway analysis based on the differentially expressed genes revealed that PB depletion increased the activation of several signaling pathways involved in the innate immune response. The proinflammatory cytokine IL-1β was significantly upregulated following infection of PB-deficient KO cells, but not in WT cells, at the mRNA and protein levels. Furthermore, the rescue of PB assembly in KO cells by GFP-tagged wild-type EDC4 (+WT) suppressed IL-1β expression, whereas KO cells with the C-terminal-deleted mutant EDC4 (+Δ) failed to rescue PB assembly and downregulate IL-1β production. Our results suggest that T. cruzi assembles host PBs to counteract antiparasitic innate immunity.

FTIR spectroscopy is well known for its molecule fingerprinting capability but is also able to differentiate classes in complex biological systems. This includes strain typing and species level identification of bacterial, yeast or fungal cells, as well as distinguishing between cell layers in eukaryotic tissues. However, its use for the identification of macromolecules such as proteins remains underexplored and rarely used in practice. Here we demonstrate the efficacy of FTIR microspectroscopy coupled with machine learning methods for rapid and accurate identification of proteins in their dry state within minutes, from very small quantities of material, if they are obtained in a pure aqueous solution. FTIR microspectroscopy can provide additional information beside identification: it can detect small differences among different purification batches potentially originating from post-translational modifications or distinct folding states. Moreover, it distinguishes glycoproteins and evaluate glycosylation while detecting contaminants. This methodology presents itself as a valuable quality control tool in protein purification processes or any process requiring the utilization of precisely identified, pure proteins.

Increased plasma levels of serum amyloid A (SAA), an acute-phase protein that is secreted in response to inflammation, may lead to the accumulation of amyloid in various organs thereby obstructing their functions. Severe cases can lead to a systemic disorder called AA amyloidosis. Previous studies suggest that the N-terminal helix is the most amyloidogenic region of SAA. Moreover, computational studies implicated a significant role for Arg-1 and the residue-specific interactions formed during the fibrillization process. With a focus on the N-terminal region of helix-1, SAA_1–13, mutational analysis was employed to interrogate the roles of the amino acid residues, Arg-1, Ser-5, Glu-9, and Asp-12. The truncated SAA_1–13 fragment was systematically modified by substituting the key residues with alanine or uncharged but structurally similar amino acids. We monitored the changes in the amyloidogenic propensities, associated conformational markers, and morphology of the amyloids resulting from the mutation of SAA_1–13. Mutating out Arg-1 resulted in much reduced aggregation propensity and a lack of detectable β-structures alluding to the importance of salt-bridge interactions involving Arg-1. Our data revealed that by systematically mutating the key amino acid residues, we can modulate the amyloidogenic propensity and alter the time-dependent conformational variation of the peptide. When the behaviors of each mutant peptide were analyzed, they provided evidence consistent with the aggregation pathway predicted by MD simulation studies. Here, we detail the important temporal molecular interactions formed by Arg-1 with Ser-5, Glu-9, and Asp-12 and discuss its mechanistic implications on the self-assembly of the helix-1 region of SAA.

Phenols and phenolic compounds are major plant metabolites used in industries to produce pesticides, dyes, medicines, and plastics. These compounds enter water bodies, soil, and living organisms via such industrial routes. Some polyphenolic compounds like phenolic acids, flavonoids have antioxidant and organoleptic qualities, as well as preventive effects against neurodegenerative illnesses, cardiovascular disease, diabetes, and cancer. However, many of the polyphenolic compounds, such as Bisphenol A, phthalates, and dioxins also cause major environmental pollution and endocrine disruption, once the dose level becomes objectionable. The development of reliable and rapid methods for studying their dose dependency, high-impact detrimental effects, and continuous monitoring of phenol levels in humans and environmental samples is a crucial necessity of the day. Enzymatic biosensors employing phenol oxidases like tyrosinase, peroxidase and laccase, utilizing electrochemical amperometric methods are innovative methods for phenol quantification. Enzymatic biosensing, being highly sensitive and efficacious technique, is illuminated in this review article as a progressive approach for phenol quantification with special emphasis on laccase amperometric biosensors. Even more, the review article discussion is extended up to nanozymes, composites of metal organic frameworks (MOFs), and molecularly imprinted polymers (MIPs) as some emerging species for electro-chemical sensing of phenols. Applications of phenol quantification and green biosensing are also specified. A concrete summary of the innovative polyphenol detection approaches with futuristic scope indicates a triumph over some existing constraints of the phenomenological approaches providing an informative aisle to the modern researchers towards the bulk readability.

Human glycosyltransferases (GTs) play crucial roles in glycan biosynthesis, exhibiting diverse domain architectures. This study explores the functional diversity of “add-on” domains within human GTs, using data from the AlphaFold Protein Structure Database. Among 215 annotated human GTs, 74 contain one or more add-on domains in addition to their catalytic domain. These domains include lectin folds, fibronectin type III, and thioredoxin-like domains and contribute to substrate specificity, oligomerization, and consequent enzymatic activity. Notably, certain GTs possess dual enzymatic functions due to catalytic add-on domains. The analysis highlights the importance of add-on domains in enzyme functionality and disease implications, such as congenital disorders of glycosylation. This comprehensive overview enhances our understanding of GT domain organization, providing insights into glycosylation mechanisms and potential therapeutic targets.

Background

Pancreatic cancer (PC) is characterized by a poor prognosis and limited treatment options. Ferroptosis plays an important role in cancer, SET and MYND domain-containing protein 2 (SMYD2) is widely expressed in various cancers. However, the role of SMYD2 in regulating ferroptosis in PC remains unexplored. This study aimed to investigate the role of SMYD2 in mediating ferroptosis and its mechanistic implications in PC progression.

Methods

The levels of SMYD2, c-Myc, and NCOA4 were assessed in PC tissues, and peritumoral tissues. SMYD2 expression was further analyzed in human PC cell lines. In BxPC3 cells, the expression of c-Myc, NCOA4, autophagy-related proteins, and mitochondrial morphology, was evaluated following transfection with si-SMYD2 and treatment with autophagy inhibitors and ferroptosis inhibitors. Ferroptosis levels were quantified using flow cytometry and ELISA assays. RNA immunoprecipitation was conducted to elucidate the interaction between c-Myc and NCOA4 mRNA. A xenograft mouse model was constructed to validate the impact of SMYD2 knockdown on PC growth.

Results

SMYD2 and c-Myc were found to be highly expressed in PC tissues, while NCOA4 showed reduced expression. Among the PC cell lines studied, BxPC3 cells exhibited the highest SMYD2 expression. SMYD2 knockdown led to decreased c-Myc levels, increased NCOA4 expression, reduced autophagy-related protein expression, mitochondrial shrinkage, and heightened ferroptosis levels. Additionally, an interaction between c-Myc and NCOA4 was identified. In vivo, SMYD2 knockdown inhibited tumor growth.

Conclusions

Targeting SMYD2 inhibits PC progression by promoting ferritinophagy-dependent ferroptosis through the c-Myc/NCOA4 axis. These findings provide insights into potential diagnostic and therapeutic strategies for PC.