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1. Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach

Author

Thea van den Bosch, Alexander P. Boichenko, Annalisa D’Urzo, Frank J. Dekker, Vincenza Andrisano, Jos Hermans, Rainer Bischoff, Chemical and Pharmaceutical Biology, Analytical Biochemistry, Biopharmaceuticals, Discovery, Design and Delivery (BDDD), Medicinal Chemistry and Bioanalysis (MCB), D'Urzo, A, Boichenko, Ap, van den Bosch, T, Hermans, J, Dekker, F, Andrisano, V, and Bischoff, R.

Subjects
0301 basic medicine, Tandem mass spectrometry, DEACETYLASE INHIBITORS, Lysine, Histone deacetylase (HDAC) inhibitors, Histone deacetylase (HDAC) inhibitor, Biochemistry, Cell Line, Analytical Chemistry, Histones, Histone H4, Mice, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, medicine, Animals, Amino Acid Sequence, Post-translation modification (PTM), Derivatization, Chromatography, High Pressure Liquid, PROPIONYLATION, Chymotrypsin, Chromatography, biology, Reproducibility of Results, Acetylation, MASS-SPECTROMETRY, MS, Trypsin, ALZHEIMERS-DISEASE, Histone Deacetylase Inhibitors, 030104 developmental biology, Histone acetylation, chemistry, Multiple reaction monitoring (MRM), biology.protein, Histone deacetylase, 030217 neurology & neurosurgery, Research Paper, medicine.drug
Abstract

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R2 ≥ 0.94), precision (RSD ≤ 10 %), and accuracy (≤27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)–K8 and K5–K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9431-1) contains supplementary material, which is available to authorized users.

Published
2016

2. Identification of Reticulocyte Binding Domain of Plasmodium ovale curtisi Duffy Binding Protein (PocDBP) Involved in Reticulocyte Invasion.

Author

Hoque, Mohammad Rafiul, Nyunt, Myat Htut, Han, Jin-Hee, Muh, Fauzi, Lee, Seong-Kyun, Park, Ji-Hoon, Lu, Feng, Park, Won Sun, Han, Eun-Taek, and Na, Sunghun

Subjects
CARRIER proteins, PLASMODIUM, RECOMBINANT proteins, AMINO acid sequence, PLASMODIUM vivax, IMMUNE serums, CHYMOTRYPSIN
Abstract

The Plasmodium ovale curtisi (Poc) prevalence has increased substantially in sub-Saharan African countries as well as regions of Southeast Asia. Poc parasite biology has not been explored much to date; in particular, the invasion mechanism of this malaria parasite remains unclear. In this study, the binding domain of the Duffy binding protein of P. ovale curtisi (PocDBP) was characterized as an important ligand for reticulocyte invasion. The homologous region of the P. vivax Duffy binding protein in PocDBP, named PocDBP-RII herein, was selected, and the recombinant PocDBP-RII protein was expressed in an Escherichia coli system. This was used to analyze reticulocyte binding activity using fluorescence-activated cell sorting and immune serum production in rabbits. The binding specificity was proven by treating reticulocytes with trypsin, chymotrypsin and neuraminidase. The amino acid sequence homology in the N-terminal Cys-rich region was found to be ~ 44% between PvDBP and PocDBP. The reticulocyte binding activity of PocDBP-RII was significantly higher than the erythrocyte binding activity and was concentration dependent. Erythrocyte binding was reduced significantly by chymotrypsin treatment and inhibited by an anti-PocDBP-RII antibody. This finding suggests that PocDBP may be an important ligand in the reticulocyte invasion process of P. ovale curtisi. [ABSTRACT FROM AUTHOR]

Published
2021
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3. Molecular insights into mechanisms of lepidopteran serine proteinase resistance to natural plant defenses

Author

Walter R. Terra and Fábio K. Tamaki

Subjects
Models, Molecular, Serine Proteinase Inhibitors, Molecular Sequence Data, Biophysics, Biochemistry, Serine, Serine proteinases, Plant defense against herbivory, medicine, Animals, Amino Acid Sequence, Molecular Biology, Peptide sequence, Plant Physiological Phenomena, Chymotrypsin, biology, Sequence Homology, Amino Acid, fungi, Active site, Cell Biology, Trypsin, LEPIDOPTERA, Lepidoptera, biology.protein, Serine Proteases, medicine.drug
Abstract

Plants have a wide range of chemical defenses against predation, including substances that target digestive serine proteinases of herbivorous. Previous works demonstrated that lepidopteran insects have digestive serine proteinases resistant to plant proteinase inhibitors (PPIs) and ketone modifications, while coleopteran ones are sensitive to those plant defenses. This paper focuses on molecular aspects that lead lepidopteran serine proteinases to PPI and ketone modification resistance. Using biochemical experiments and computer 3D modeling we demonstrated that lepidopteran trypsins are more hydrophobic than coleopteran ones, a feature associated to trypsin oligomerization and decreased inhibition by PPI. Moreover, the determination of pKa values of chymotrypsin catalytic residues obtained by TPCK modification indicates that the environment around the active site of ketone-resistant and –sensitive chymotrypsins are different. Structural analysis using resistant and sensitive chymotrypsins data allowed us to point 2 hotspot regions around the active site that could explain the observed differences. Our set of results highlights features of serine proteinases important for understanding the resistance of insects to plant chemical defenses.

Published
2015