bims-hypusi 2025-05-25 papers

Issue of 2025–05–25
two papers selected by
Sebastian J. Hofer, Max Delbrück Center

Methods Enzymol. 2025 ;pii: S0076-6879(25)00140-5. [Epub ahead of print]715 1-17

Genetic analyses of Myc and hypusine circuits in tumorigenesis.

A prominent metabolic pathway induced by MYC family oncoproteins in cancer is the polyamine-hypusine circuit, which post-translationally modifies a specific lysine residue of eukaryotic translation initiation factor 5 A (eIF5A) with a unique amino acid coined hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. This modification occurs in a two-step process, whereby the aminobutyl group of the polyamine spermidine is covalently linked to lysine-50 of eIF5A via deoxyhypusine synthase (DHPS) to form the intermediate deoxyhypusinated eIF5A, which is subsequently hydoxylated by deoxyhypusine hydroxylase (DOHH) to form the fully mature eIF5AHyp. As a result, eIF5AHyp is elevated in MYC-driven cancers. Recently it has become evident that eIF5AHyp (i) plays key roles in the development, progression and maintenance of tumors; and (ii) eIF5AHyp functions are often tissue/cell context-specific. Thus, it is important to mechanistically assess how eIF5AHyp affects normal cells and tumorigenesis using suitable in vivo and ex vivo models. In this chapter, we describe the methods used in our laboratory to assess the effects of MYC-polyamine-hypusine axis on the development and maintenance of MYC-driven B-cell lymphoma. The goals of this chapter are twofold. First, we discuss genetic and cell biological approaches that can be applied to assess roles of eIF5AHyp on lymphoma and normal B cell development. Second, we discuss methods that can be used to assess the roles of eIF5AHyp on the growth and maintenance of lymphoma. Collectively, these approaches provide a template that can be applied to evaluate roles of any putative regulator of the development and/or maintenance of lymphoma.

Keywords: B-cell; DHPS; Eμ-Myc; Hypusine; Lymphoma; Myc; ODC; Polyamines; eIF5A

DOI: https://doi.org/10.1016/bs.mie.2025.02.005

Methods Enzymol. 2025 ;pii: S0076-6879(25)00118-1. [Epub ahead of print]715 155-182

Yeast reconstituted translation assays for analysis of eIF5A function.

Byung-Sik Shin, Thomas E Dever.

Polyamines are critically important for protein synthesis. Through their positive ionic charge, polyamines readily bind to ribosomes, as well as to mRNAs and tRNAs. Moreover, the polyamine spermidine serves as a substrate for the synthesis of hypusine, an essential post-translational modification on the translation factor eIF5A. Though originally thought to function in translation initiation, eIF5A is now known to generally promote translation elongation and termination. Moreover, translation of certain motifs like polyproline show a greater dependency on eIF5A. In this chapter, we describe the biochemical assays we use to study eIF5A and its regulation. Owing to the complex nature of protein synthesis, these assays require the purification of over 10 translation factors plus ribosomes, tRNAs, and aminoacyl-tRNA synthetases. We describe the methods used to purify these components, to synthesize the mRNA templates for translation, and to resolve the translation products by electrophoretic thin-layer chromatography. With the recent identification of eIF5A as a key target for regulating the synthesis of polyamine synthesis and transport, and the recent identification of mutations in eIF5A causing a neurodevelopmental disorder, the assays described in this chapter will be useful in further elucidating the function and regulation of this enigmatic protein.

Keywords: DHPS; DOHH; Elongation; Hypusine; Reconstituted translation; Ribosome; Termination; Yeast; eIF5A

DOI: https://doi.org/10.1016/bs.mie.2025.01.076