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Molecules (Basel, Switzerland) May 2024The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial...
The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of () were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.
Topics: Aryldialkylphosphatase; Humans; Proteomics; Protein Refolding; Pseudomonas aeruginosa; Enzyme Stability; Biofilms; Protein Processing, Post-Translational
PubMed: 38893310
DOI: 10.3390/molecules29112434 -
International Journal of Molecular... Jun 2024(maize) is a staple food, feed, and industrial crop. Heat stress is one of the major stresses affecting maize production and is usually accompanied by other stresses,...
(maize) is a staple food, feed, and industrial crop. Heat stress is one of the major stresses affecting maize production and is usually accompanied by other stresses, such as drought. Our previous study identified a heterotrimer complex, ZmNF-YA1-YB16-YC17, in maize. ZmNF-YA1 and ZmNF-YB16 were positive regulators of the drought stress response and were involved in maize root development. In this study, we investigated whether ZmNF-YA1 confers heat stress tolerance in maize. The mutant and overexpression lines were used to test the role of ZmNF-YA1 in maize thermotolerance. The mutant was more temperature-sensitive than the wild-type (WT), while the overexpression lines showed a thermotolerant phenotype. Higher malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation were observed in the mutant, followed by WT and overexpression lines after heat stress treatment, while an opposite trend was observed for chlorophyll content. RNA-seq was used to analyze transcriptome changes in and its wild-type control W22 in response to heat stress. Based on their expression profiles, the heat stress response-related differentially expressed genes (DEGs) in compared to WT were grouped into seven clusters via -means clustering. Gene Ontology (GO) enrichment analysis of the DEGs in different clades was performed to elucidate the roles of ZmNF-YA1-mediated transcriptional regulation and their contribution to maize thermotolerance. The loss function of led to the failure induction of DEGs in GO terms of protein refolding, protein stabilization, and GO terms for various stress responses. Thus, the contribution of ZmNF-YA1 to protein stabilization, refolding, and regulation of abscisic acid (ABA), ROS, and heat/temperature signaling may be the major reason why overexpression enhanced heat tolerance, and the mutant showed a heat-sensitive phenotype.
Topics: Zea mays; Heat-Shock Response; Thermotolerance; Gene Expression Regulation, Plant; Plant Proteins; Reactive Oxygen Species; Mutation; CCAAT-Binding Factor; Gene Expression Profiling; Transcriptome; Plants, Genetically Modified
PubMed: 38892463
DOI: 10.3390/ijms25116275 -
IScience Jun 2024Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to...
Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.
PubMed: 38868206
DOI: 10.1016/j.isci.2024.110009 -
Experimental Cell Research Jun 2024Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause...
Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of β-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.
PubMed: 38852763
DOI: 10.1016/j.yexcr.2024.114118 -
ELife Jun 2024CRISPR prime editing () requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (), an extended version of a standard...
CRISPR prime editing () requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (), an extended version of a standard guide RNA () that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as sixfold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.
Topics: Zebrafish; Animals; Gene Editing; RNA, Guide, CRISPR-Cas Systems; CRISPR-Cas Systems; CRISPR-Associated Protein 9; Embryo, Nonmammalian; RNA Folding
PubMed: 38847802
DOI: 10.7554/eLife.90948 -
Plant, Cell & Environment Jun 2024Wheat (Triticum aestivum L.) is an important cereal crop cultivated and consumed worldwide. Global warming-induced escalation of temperature during the seedling and...
Wheat (Triticum aestivum L.) is an important cereal crop cultivated and consumed worldwide. Global warming-induced escalation of temperature during the seedling and grain-filling phase adversely affects productivity. To survive under elevated temperatures, most crop plants develop natural mechanisms at molecular level by activating heat shock proteins. However, other heat stress-related proteins like heat acclimatization (HA) proteins are documented in hexaploid wheat but have not been explored in detail in its diploid and tetraploid progenitors, which might help to overcome elevated temperature regimes for short periods. Our study aims to explore the potential HA genes in progenitors Triticum durum and Aegilops tauschii that perform well at higher temperatures. Seven genes were identified and phylogenetically classified into three families: K homology (KH), Chloroplast protein-enhancing stress tolerance (CEST), and heat-stress-associated 32 kDa (HSA32). Protein-protein interaction network revealed partner proteins that aid mRNA translation, protein refolding, and reactive species detoxification. Syntenic analysis displayed highly conserved relationships. RT-qPCR-based expression profiling revealed HA genes to exhibit diverse and dynamic patterns under high-temperature regimes, suggesting their critical role in providing tolerance to heat stress. The present study furnishes genetic landscape of HA genes that might help in developing climate-resilient wheat with higher acclimatization potential.
PubMed: 38847343
DOI: 10.1111/pce.14992 -
Gene Oct 2024The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal...
The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal rings, binds to around 10 % of newly synthesized polypeptides and facilitates their folding in ATP dependent manner. In Leishmania, among five subunits of TCP1 complex, identified either by transcriptome or by proteome analysis, only LdTCP1γ has been well characterized. It forms biologically active homo-oligomeric complex and plays role in protein folding and parasite survival. Lack of information regarding rest of the TCP1 subunits and its structural configuration laid down the necessity to study individual subunits and their role in parasite pathogenicity. The present study involves the cloning, expression and biochemical characterization of TCP1ε subunit (LdTCP1ε) of Leishmania donovani, the causative agent of visceral leishmaniasis. LdTCP1ε exhibited significant difference in primary structure as compared to LdTCP1γ and was evolutionary close to LdTCP1 zeta subunit. Recombinant protein (rLdTCP1ε) exhibited two major bands of 132 kDa and 240 kDa on native-PAGE that corresponds to the dimeric and tetrameric assembly of the epsilon subunit, which showed the chaperonin activity (ATPase and luciferase refolding activity). LdTCP1ε also displayed an increased expression upto 2.7- and 1.8-fold in the late log phase and stationary phase promastigotes and exhibited majorly vesicular localization. The study, thus for the first time, provides an insight for the presence of highly diverge but functionally active dimeric/tetrameric TCP1 epsilon subunit in Leishmania parasite.
Topics: Leishmania donovani; Chaperonin Containing TCP-1; Protozoan Proteins; Protein Multimerization; Recombinant Proteins; Protein Subunits; Cloning, Molecular; Amino Acid Sequence; Chaperonins; Protein Folding
PubMed: 38844270
DOI: 10.1016/j.gene.2024.148637 -
Journal of Colloid and Interface Science May 2024Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded...
HYPOTHESIS
Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded state. However, further studies are required to refine our understanding of the underlying mechanism of the refolding process. While interactions between nonionic surfactants and tightly folded proteins are not anticipated, we hypothesized that intermediate stages of surfactant-induced unfolding could define new interaction mechanisms by which nonionic surfactants can further alter protein conformation.
EXPERIMENTS
In this work, the behavior of three model proteins (human growth hormone, bovine serum albumin, and β-lactoglobulin) was investigated in the presence of the anionic surfactant sodium dodecylsulfate, the nonionic surfactant β-dodecylmaltoside, and mixtures of both surfactants. The transitions occurring to the proteins were determined using intrinsic fluorescence spectroscopy and far-UV circular dichroism. Based on these results, we developed a detailed interaction model for human growth hormone. Using nuclear magnetic resonance and contrast-variation small-angle neutron scattering, we studied the amino acid environment and the conformational state of the protein.
FINDINGS
The results demonstrate the key role of surfactant cooperation in defining the conformational state of the proteins, which can shift away or toward the folded state depending on the nonionic-to-ionic surfactant ratio. Dodecylmaltoside, initially a non-interacting surfactant, can unexpectedly associate with sodium dodecylsulfate-unfolded proteins to further impact their conformation at low nonionic-to-ionic surfactant ratio. When this ratio increases, the protein begins to retrieve the folded state. However, the native conformation cannot be fully recovered due to remnant surfactant molecules still adsorbed to the protein. This study demonstrates that the conformational landscape of the protein depends on a delicate interplay between the surfactants, ultimately controlled by the ratio between them, resulting in unpredictable changes in the protein conformation.
PubMed: 38838632
DOI: 10.1016/j.jcis.2024.05.157 -
International Journal of Biological... Jun 2024The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae...
Changes in physicochemical, structural and functional properties, and lysinoalanine formation during the unfolding and refolding of pH-shifted black soldier fly larvae albumin.
The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH shift, pH shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH shift treatment. In addition, pH shift modified BSFLA tended to form β-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH shifting did not cause irreversible denaturation of protein molecules. Therefore, pH-shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food.
Topics: Animals; Hydrogen-Ion Concentration; Protein Unfolding; Lysinoalanine; Hydrophobic and Hydrophilic Interactions; Larva; Albumins; Diptera; Solubility; Protein Refolding; Insect Proteins; Chemical Phenomena
PubMed: 38825263
DOI: 10.1016/j.ijbiomac.2024.132801 -
Chemical Science May 2024Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS). More efficient ligation methods that circumvent the...
Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS). More efficient ligation methods that circumvent the requirement of a peptidyl thioester and thiol additive-which allow the following desulfurization or refolding in one pot-are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl -acyl pyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester or thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin an N-to-C sequential ligation-MFD strategy, the semi-synthesis of the copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.
PubMed: 38817582
DOI: 10.1039/d3sc06697e