scholarly journals Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

2020 ◽  
Author(s):  
Manish Bhattacharjee ◽  
Navin Adhikari ◽  
Renu Sudhakar ◽  
Zeba Rizvi ◽  
Divya Das ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasmodium Falciparum ◽  
Western Blot ◽  
Wild Type ◽  
Malaria Parasites ◽  
Post Translational Modifications ◽  
Functional Conservation ◽  
Cellular Processes ◽  
Physiological Substrates

ABSTRACTA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (Δrub1) or NEDD8 conjugating E2 enzyme (ΔUbc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

scholarly journals Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Manish Bhattacharjee ◽  
Navin Adhikari ◽  
Renu Sudhakar ◽  
Zeba Rizvi ◽  
Divya Das ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Target ◽  
Parasite Development ◽  
Wild Type ◽  
Post Translational Modifications ◽  
Apicomplexan Parasites ◽  
Cellular Processes ◽  
Ring E3 ◽  
Physiological Substrates

AbstractA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.

scholarly journals Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1701
Author(s):  
Lorenz Weidenauer ◽  
Manfredo Quadroni
Keyword(s):  
Mass Spectrometry ◽  
K562 Cells ◽  
Phosphorylation Sites ◽  
Activity Regulation ◽  
Wild Type ◽  
Post Translational Modifications ◽  
Linker Region ◽  
Cellular Processes ◽  
Tryptic Cleavage ◽  
Client Proteins

Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone.

scholarly journals Integrating bioinformatics tools to investigate protein phosphorylation

2015 ◽  
Author(s):  
Dimitrios Vlachakis ◽  
Elena Bencurova ◽  
Mangesh Bhide ◽  
Sophia Kossida
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Benchmark Study

Protein phosphorylation is one of the most important protein post-translational modifications and plays a role in numerous cellular processes including recognition, signaling and degradation. It can be studied experimentally by various methodologies, like employing western blot analysis, site-directed mutagenesis, 2 D gel electrophoresis, mass spectrometry etc. A number of in silico tools have also been developed in order to predict plausible phosphorylation sites in a given protein. In this review, we conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. The first place was taken by GPS 2.2, having predicted all phosphorylation sites with a 83% fidelity while in second place came NetPhos 2.0 with 69%.

scholarly journals Integrating bioinformatics tools to investigate protein phosphorylation

2015 ◽  
Author(s):  
Dimitrios Vlachakis ◽  
Elena Bencurova ◽  
Mangesh Bhide ◽  
Sophia Kossida
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Benchmark Study

Protein phosphorylation is one of the most important protein post-translational modifications and plays a role in numerous cellular processes including recognition, signaling and degradation. It can be studied experimentally by various methodologies, like employing western blot analysis, site-directed mutagenesis, 2 D gel electrophoresis, mass spectrometry etc. A number of in silico tools have also been developed in order to predict plausible phosphorylation sites in a given protein. In this review, we conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. The first place was taken by GPS 2.2, having predicted all phosphorylation sites with a 83% fidelity while in second place came NetPhos 2.0 with 69%.

scholarly journals Molecular Characterization of the CytochromebGene andIn VitroAtovaquone Susceptibility of Plasmodium falciparum Isolates from Kenya

2015 ◽  
Vol 59 (3) ◽  
pp. 1818-1821 ◽  
Author(s):  
Luicer A. Ingasia ◽  
Hoseah M. Akala ◽  
Mabel O. Imbuga ◽  
Benjamin H. Opot ◽  
Fredrick L. Eyase ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Polymorphism ◽  
Molecular Characterization ◽  
Mutant Allele ◽  
Inhibitory Concentration ◽  
Wild Type Allele ◽  
Wild Type ◽  
Western Kenya ◽  
Content Type

ABSTRACTThe prevalence of a genetic polymorphism(s) at codon 268 in the cytochromebgene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227Plasmodium falciparumparasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.

scholarly journals Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 207 ◽  
Author(s):  
Stephen R. Johnson ◽  
Hillary G. Rikli
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Protein Protein Interactions ◽  
High Definition ◽  
Vast Number ◽  
Post Translational Modifications ◽  
C Terminus ◽  
Acid Isomerization

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

Characterization of a Plasmodium falciparum PHISTc protein, PF3D7_0801000, in blood- stage malaria parasites

2021 ◽  
Vol 80 ◽  
pp. 102240
Author(s):  
Hikaru Nagaoka ◽  
Bernard N. Kanoi ◽  
Masayuki Morita ◽  
Takahiro Nakata ◽  
Nirianne M.Q. Palacpac ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Stage ◽  
Malaria Parasites ◽  
Blood Stage Malaria

scholarly journals Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein β antibody in differentiating myoblasts

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Hamood AlSudais ◽  
Nadine Wiper-Bergeron
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Transcription Factors ◽  
Western Blot ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Intense Band ◽  
Cellular Processes ◽  
Enhancer Binding Protein ◽  
Myoblast Cells

Abstract Objective CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPβ expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPβ, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPβ, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPβ, the band was present when C/EBPβ was knocked down, confirming specificity for a protein other than C/EBPβ. The objective of this work was to identify the contaminating reactivity. Results We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPβ is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPβ isoforms.

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