Research Interests
The role of LINE1-derived pathogen associated molecular patterns in senescence and inflammaging
Transposable Elements (TEs), also known as "jumping genes", are defined as mobile DNA elements that can replicate and intergrate themselves into different locations within the host genome 1. TEs have been identified in all organisms and often occupy a large proportion of their host genome 2. TEs are divided into two classes based upon their mechanism of transposition. Class I TEs, (a.k.a retrotransposable elements; RTEs), mobilize via a ‘copy-and-paste’ mechanism, where an RNA intermediate is reverse-transcribed into cDNA that is subsequently inserted in a different location within the host genome 3. Class II TEs, (a.k.a. DNA transposons), proliferate through a DNA intermediate, in a more direct way, through a ‘cut-and-paste’ mechanism 4,5. The most abundant RTEs in the human genome are LINE-1 (L1) and Alu elements, accounting for ~30% of the genome6. Unlike Alu, L1s encode their own proteins and are therefore capable of autonomous retrotransposition. In more detail, L1 consists of a 5’- UTR with an internal promoter, two open reading frames (ORFs), namely ORF1, ORF2 and a 3’-UTR with weak cleavage and polyadenylation signal 7–9. ORF1 encodes for the ORF1p (~40 kDa), which forms a homotrimer and is a putative nucleic acid chaperone 10,11, whereas ORF2 encodes for the ORF2p (~150 kDa), a multidomain enzyme with both endonuclease (EN) and reverse transcriptase (RT) activities 12,13. L1 ORF1p and ORF2p preferentially assemble with their own encoding RNA (cis preference), forming ribonucleoprotein complexes (RNPs), presumably near or at the site of translation 14,15. Notably, at all stages of the L1 retrotransposition process (i.e., L1’s ‘life cycle’), L1s engage various host-encoded proteins, some facilitating and others impeding its action 16, 17, 18, 19,20,21, 22, 23.
L1 retrotransposition proceeds through DNA/RNA/protein-containing intermediates across cellular compartments, predominantly in the nucleus and cytoplasm (Fig. 1). L1 enters the nucleus and gains access to chromatin as the cell starts to divide and the nuclear membrane breaks down 24. According to the most recent model for L1 proliferation (termed: target-primed reverse transcription; TPRT) 25,26, cDNA production by ORF2p takes place in the nucleus and is primed by a free 3’ hydroxyl group, generated by ORF2p EN cleavage of host DNA. In healthy cells, L1 transcription is suppressed both epigenetically and by p53 and other transcription factors 27–33. However, in cell senescence, L1s become activated and trigger a type-I interferon (IFN-I) response, causing inflammation 34. Additionally, cell senescence is characterized by cell proliferation arrest, as well as by abnormal ORF2p-driven production of cytoplasmic cDNAs and DNA:RNA hybrids. Consequently, L1 RNPs become trapped and accumulate in the cytoplasm, while the hybrids produced act as pathogen associated molecular patterns (PAMPs) that activate host cytoplasmic pattern recognition receptors (PRRs), including the cGAS/STING DNA sensing pathway 35–37. As a result, this induces an IFN-I response, leading to increased secretion of proinflammatory cytokines, known as the senescence associated secretory phenotype (SASP) 34. L1 RNPs are highly heterogeneous in composition and apart from DNA:RNA hybrids they also include other PAMPs. For example, L1 RNAs are enriched in CpGs motifs and therefore are themselves PAMPs, but also assemble with dsRNA-forming Alus 38,39 . Therefore, L1 RNPs can act as sites of aggregation of cytoplasmic hybrids and other PAMPs. Collectively, stimulation of innate immunity in response to RTE nucleic acids (NAs), especially by ORF2p-derived cDNAs and hybrids constitutes a novel mechanism for understanding senescence associated diseases. L1 RNPs assemble several pathogenic NAs, but their relative contributions to host innate immune responses remain uncharacterised. We aim to characterize the composition of L1 RNPs in cell senescence and dissect the immune pathways associated with RTE-derived NAs, in particular cDNAs, dsRNAs and DNA:RNA hybrids generated by aberrant cytoplasmic L1 activity.
References
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Dissecting the molecular features of a rare pediatric cancer, fibrolamellar carcinoma, using a double-barreled interactome mapping approach.
Fibrolamellar carcinoma (FLC) is a rare and lethal subtype of liver cancer with a global incidence of 0.02 per 100,000 per year1. However, this tumor primarily affects adolescent and pediatric patients without a history of liver disease, making it a devastatingly impactful disease despite its comparative rarity. The mainstay of treatment is complete surgical removal, due to its refractory nature against potential cures such as chemoradiation 2. FLCs lack obvious symptoms or serum tumor markers; metastasis at presentation is frequent and often progressive and therefore fatal in nature 1. A specific, diagnostic genetic fusion event was identified as a candidate driver of FLC - DNAJB1::PRKACA 3. A deletion of 400 kb in chromosome 19 results in the fusion of fragments of the genes encoding the heat shock protein DNAJ (Hsp40), subfamily B, member 1, (DNAJB1) and the catalytic subunit alpha of protein kinase A (PRKACA) 3. The deletion results in a chimeric RNA transcript, in which exon 1 of DNAJB1 replaces exon 1 of PRKACA, producing a fusion protein in which the J-domain of DNAJB1 (a.k.a. HSP40) replaces an amino-terminal segment of the catalytic subunit alpha of the cyclic AMP-dependent protein kinase A (a.k.a. PKA Cα) 4, referred to as DP hereafter (diagrammed in Fig. 1). Notably, DP retains kinase activity and has been shown to have tumorigenic properties not explainable by simple overexpression of PRKACA 5.
While the DP gene fusion event was previously thought to be unique to FLC, recent studies have also identified other pancreatobiliary neoplasms that exhibit the same defect (and others) 6, 7, underscoring the importance and health relevance of studying the roles of rare kinase fusion events in hepatopancreatobiliary tumors, and the role of this specific fusion event in FLC. We aim to conduct a comparative analysis of the protein interactions and phosphorylation patterns exhibited by wild-type PRKACA, DNAJB1, and DP fusion proteins, comparing what is observed in normal liver, liver adenoma, hepatocellular carcinoma, and FLC tissues.
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3. Honeyman, J. N. et al. Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma. Science 343, 1010–1014 (2014).
4. Liu, C., Ke, P., Zhang, J., Zhang, X. & Chen, X. Protein kinase inhibitor peptide as a tool to specifically inhibit protein kinase A. Front. Physiol. 11, 574030 (2020).
5. Kastenhuber, E. R. et al. DNAJB1-PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma. Proc Natl Acad Sci USA 114, 13076–13084 (2017).
6. Zhu, C. et al. The fusion landscape of hepatocellular carcinoma. Mol. Oncol. 13, 1214–1225 (2019).
7. Vyas, M. et al. DNAJB1-PRKACA fusions occur in oncocytic pancreatic and biliary neoplasms and are not specific for fibrolamellar hepatocellular carcinoma. Mod. Pathol. 33, 648–656 (2020).
