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Regioselective Protein Oxidative Cleavage Enabled by an Enzyme-like Recognition of an Inorganic Metal Oxo Cluster Ligand

ABSTRACT: Metal-catalyzed oxidation reactions (MCO) is one of the most applicable methods to induce protein oxidative modifications (e.g., protein oxidation, protein oxidative cleavage and crosslinking), which are essential techniques for many applications in biotechnology and redox proteomics. However, a suitable ligand is needed to assure high stability and enhance the metal catalytic efficiency in order to induce these oxidative modifications in a selective manner. Herein, we report the influence of the polyoxometalates (POMs) as inorganic ligands on the catalytic efficiency of Cu ions to induce oxidative cleavage of a protein. In the presence of ascorbic acid as (Asc), the Cu-substituted POM (Cu-POM), α2-K8P2W17O61(Cu2+.OH2), (CuWD), cleaved hen egg white lysozyme (HEWL), a protein consisting of 129 amino acids, producing only four peptide fragments. Analyzing the intact protein, in absence and presence of CuWD/Asc, and the peptide four peptide fragments via nLC-MS/MS revealed that CuWD/Asc induced oxidative modifications and cleavage at and/or near CuWD/HEWL binding sites

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Gallus Gallus (chicken)

TISSUE(S): Egg

SUBMITTER: Tamara Vasovic

LAB HEAD: Tanja Ćirković Veličković

PROVIDER: PXD037983 | Pride | 2023-01-30

REPOSITORIES: Pride

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Project description:MTF1 is a highly conserved metal-binding transcription factor in eukaryotes. MTF1 binds to DNA sequence motifs, termed metal response elements (MREs) to induce the expression of genes involved in metal and oxidative stress homeostasis. MTF1 is responsive to both metal excess and deprivation, and can also protect cells from oxidative and hypoxic stresses. Disruption of metal homeostasis leads to the development of several pathological states. Despite its roles in these processes , MTF1 has been shown to be required for developmental processes such as embryonic liver formation. In this study, we used multiple strategies to understand the mechanism by which MTF1 functions in skeletal muscle differentiation and to determine the role cellular copper (Cu) status plays in this process. We provide the functional relationships between MTF1, Cu, myogenic gene promoters, and MyoD, an specific component of the myogenic transcriptional machinery in differentiating primary myoblasts derived from mouse satellite cells. We found that MTF1 is induced and translocated to the nucleus upon initiation of myogenesis. Consistent with previous studies from our laboratory , addition of non-toxic concentrations of Cu promote myogenesis and enhanced MTF1 expression. CRISPR/Cas9-mediated depletion of MTF1 demonstrated that MTF1 is essential for proper development of skeletal muscle, as partial Mtf1 knockdown leads to apoptosis to differentiating myoblasts. MTF1 was also found to bind Cu at a carboxy-terminal tetra-cysteine cluster, which may contribute to the mobilization of Cu to the nucleus during myogenesis. ChIP-seq and ChIP-qPCR analyses showed that MTF1 binds at the promoter regions of myogenic genes as part of a complex with MyoD, the master transcriptional regulator of the myogenic lineage. These results have the potential to initiate a new area of research in MTF1 function and in the regulation of myogenesis. Furthermore, these studies set the basis to understand the role of Cu at the transcriptional level, affects growth and development and will contribute to the largely unexplored are of muscular phenotypes observed in human pathologies associated to Cu misbalance, such as Menkes’ and Wilson’s diseases.

Project description:Human ingestion of microplastics (MPs) is common and inevitable due to the widespread contamination of food items, but implications on the gastric digestion of food proteins are still unknown. In this study, the interaction between pepsin and polystyrene (PS) MPs was uncovered by investigating its effect on enzyme activity and conformation while in a simulated human gastric environment. The impact on food digestion was also determined by monitoring the kinetics of protein hydrolysis through static in vitro gastric digestion of cow’s milk contaminated with PS. The binding of pepsin to PS showed that surface chemistry of MPs dictates binding affinity. The key contributor to pepsin adsorption seems to be π−π interactions between the aromatic residues and the PS phenyl rings. During quick exposure (10 minutes) of pepsin to increasing concentrations (222, 2219, 22188 particles/mL) of 10 μm PS (PS10) and 100 μm PS (PS100), total enzymatic activities were not affected remarkably. However, upon prolonged exposure at 1 and 2 hours, preferential binding of pepsin to the small, low zeta-potential PS caused structural changes in the protein which led to a significant reduction of its activity. Digestion of cow’s milk mixed with PS10 resulted in transient accumulation of larger peptides (10-35 kDa) and reduced bioavailability of short peptides (2-9 kDa) in the gastric phase. This, however, was only observed at extremely high PS10 concentration (0.3 mg/mL or 5.46E+05 particles/mL). The digestion of milk peptides, bound preferentially over pepsin on the hard corona around PS10, was delayed up to 15 minutes in comparison to bulk protein digestion. Intact caseins, otherwise rapidly digested, remained bound to PS10 in hard corona for up to 15 minutes. This work presents valuable insights regarding the interaction of MPs, food proteins, and pepsin, and their dynamics during gastric digestion which provides new knowledge and understanding on the potential risks of MPs to human health.

			Action	DRS
	Ctrl_OE0087.raw	Raw
	Ctrl_peptides_1_1_0.mzid.gz	Mzid
	F1_OE0076.raw	Raw
	F1_peptides_1_1_0.mzid.gz	Mzid
	F2_OE0077.raw	Raw

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Regioselective Protein Oxidative Cleavage Enabled by an Enzyme-like Recognition of an Inorganic Metal Oxo Cluster Ligand

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