Post-Transcriptional Regulation of the Extracellular Matrix by eIF5A in Hepatic Fibrogenesis

Translation elongation is a critical, well-regulated layer in the control of protein abundance and cellular homeostasis. This study focused on the eukaryotic initiation factor 5A (eIF5A), a highly conserved translation elongation factor. Most research on eIF5A has been conducted in yeast, where it was found to promote efficient peptide-bond formation at difficult-to-translate motifs, particularly polyproline stretches. This process requires the unique hypusine modification of eIF5A by the enzymes DHPS and DOHH. eIF5A, DHPS and DOHH are upregulated in several cancers, including hepatocellular carcinoma (HCC). Metabolic HCC develops in a background of chronic inflammation and fibrosis, in which activation of hepatic stellate cells (HSCs) drives the deposition of extracellular matrix (ECM) proteins. ECM proteins, including collagens, are enriched in proline-rich motifs, which have previously been associated with eIF5A-dependent translation elongation. Inhibition of eIF5A hypusination has been shown to reduce collagen expression, although underlying mechanisms remain incompletely understood. In this thesis, the role of eIF5A and its hypusine modification was investigated in human HSC and HCC models using RNA sequencing, ribosome profiling and proteomics. TGF-β1-activated LX-2 cells were used as a fibrogenic model characterised by increased expression of ECM and ECM-associated proteins. Inhibition of hypusination with the DHPS inhibitor GC7 reduced fibrotic proteins, including collagens, despite relatively limited changes in mRNA levels. Additionally, GC7 induced a mitochondrial phenotype characterised by reduced proteins involved in mitochondrial translation and decreased mitochondrial DNA content. Responses to GC7 treatment differed between LX-2 and the HCC cell line SNU-182, supporting context-dependent regulation across HSC- and HCC-derived systems, potentially reflecting differences in TGF-β1 responsiveness. To distinguish pharmacological GC7 effects from genetic perturbation of the hypusine pathway, inducible knockdown systems targeting DHPS and eIF5A were generated. In DHPS and EIF5A knockdown, a partially overlapping ECM downregulation was observed, but it did not fully recapitulate the mitochondrial phenotype induced by GC7 treatment. The divergence between protein and RNA levels across perturbations supported predominantly post-transcriptional regulation of ECM-associated proteins. Tripeptide enrichment and ribosome profiling analyses revealed selective enrichment of motifs previously reported to require eIF5A for efficient translation, suggesting some parallels to canonical yeast models. However, no strong ribosome-stalling signatures were detected in regulated genes. Still, increased dwell times of prolines at the E and P-sites of the ribosome were observed upon eIF5A perturbation. In parallel, many collagen transcripts showed reduced translation efficiency, supporting a model in which impaired elongation feeds back on translation initia