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Identification of interacting proteins with the NP protein of the non-oncolytic H-NP366-489I strain and the non-oncolytic Herts/33 strain by mass spectrometry

ABSTRACT: Newcastle disease virus (NDV) is one of the most significant threats to the poultry industry, and it also exhibits oncolytic properties. In our research, we have identified a non-oncolytic strain, the genotype VII NDV strain I4. We have found that the lack of oncolytic characteristics in this strain is attributed to amino acids 366-489 in the NP protein. To delve into the mechanism of NP-mediated NDV oncolysis, we initiated further investigations using the highly oncolytic Herts/33 strain's NP and a recombinant NP protein with amino acids 366-489 from the I4 strain substituted in (H-NP366-489I). These proteins represent the interacting partners in the proteomics analysis of NP proteins from both viral strains. After infecting HeLa cells with each of these strains, immunoprecipitation was performed using antibodies targeting the NP protein. Since the H-NP366-489I virus produces fewer proteins during replication, we additionally overexpressed the H-NP366-489I and Herts/33 NP proteins separately for immunoprecipitation experiments.

ORGANISM(S): Homo Sapiens

SUBMITTER: Xiufan Liu

PROVIDER: PXD045334 | iProX | Tue Sep 12 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:The growth of E. coli is inhibited by millimolar concentrations of L-homoserine, which may be problematic for the industrial production of this compound or for products derived from it. In this work, an adapted laboratory evolution (ALE) was applied, which resulted in the isolation of an E. coli strain at least 10-fold more tolerant to L-homoserine than the original (MG1655) strain. Only four genomic modifications were identified after genome sequencing of this evolved strain (designed 4E), including a 49 bp truncation starting from the codon stop of thrL and leading to a modified thrL locus carrying a thrL* allele encoding a 30 amino acids polypeptide, which is 9 amino acids more than the leader peptide encoded by thrL. Replacement of thrL with thrL* enabled the initial strain to be as or slightly more tolerant to L-homoserine than the evolved 4E strain, which is explained by the rapid metabolization of L-homoserine to threonine resulting from ThrL*-dependent transcriptional activation of the threonine thrABC operon. Interestingly, in 4E strain, L-homoserine degradation went beyond threonine degradation, as tdh and kbl encoding threonine degradation pathway II and genes of the glycine cleavage system were strongly upregulated. To infer about the toxicity of L-homoserine, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which identified a potent repression of locomotion-motility-chemotaxis process. Since the magnitude of this repression was reduced in a ΔthrL mutant concomitantly with a twofold lower sensitivity of this mutant to L-homoserine, one could argue that the repression of this biological process contributed to growth inhibition by L-homoserine. Furthermore, in both wild type and thrl mutant, a strong repression of the branched-chain amino acids synthesis and transport, as well as activation of the sulphate assimilation pathway process to cysteine synthesis were observed in the presence of L-homoserine, which may also contribute to toxic effect of this compound. How this non-canonical amino acid triggers these transcriptomic changes is discussed.

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Identification of interacting proteins with the NP protein of the non-oncolytic H-NP366-489I strain and the non-oncolytic Herts/33 strain by mass spectrometry

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