Molecular Biology Master theses submitted in 2019

Hallvard Zapffe, Ingvild Aukrust, Bjørn Ivar Haukanes og Per Morten Knappskog

Functional investigations of the human E3 ubiquitin ligase HUWE1 and two HUWE1 variants associated with intellectual disability

Ubiquitination is a post-translational modification process leading to the covalent attachment of ubiquitin to lysine residue(s) in target proteins. The process involves a cascade of enzymes i.e. E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase). HUWE1 is a gene located at chromosome Xp11.22 encoding a large multi-domain protein in the HECT family of E3 ubiquitin ligases named HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1). The Homologous to the E6AP Carboxyl Terminus (HECT) domain is critical for the ubiquitination activity as it contains a conserved cysteine residue required for the ubiquitin transfer from E2 to the substrate. The ubiquitin-associated domain (UBA) plays a role in the localization of ubiquitinated proteins bound to the 26-S proteasome, while the WWE domain recognizes substrate targets for ubiquitination as well as mediate protein-protein interactions. The function(s) of the remaining three domains of unknown function (DUF908, DUF913 and DUF4414) is at the present unknown.HUWE1 is mainly involved in targeting proteins for proteasomal degradation. Pathogenic variants in HUWE1 can cause X-linked intellectual disabilities and most of those are located in the HECT domain. Therefore, most studies have so far focused on studying this domain.The overall objective of this study was to create tools for future studies of the functional properties of HUWE1 and to investigate two HUWE1 pathogenic variants Arg110Gln (located in the DUF908 domain) and Asn4075Lys (located in the HECT domain) found in two patients with intellectual disabilities. The subcloning of two different constructs encoding either i) the N-terminal DUF908 and DUF913 and ii) the C-terminal HECT domain was successful, and Arg110Gln and Asn4075Lys was made by site-directed mutagenesis in the DUF domains and HECT domain construct, respectively. The separate domains (WT and variants) were expressed in HEK 293 cells. These results could indicate a slight increase and decrease in the expression of Arg110Gln and Asn4075Lys, respectively, compared to WT. A ubiquitination assay was performed for Asn4075Lys compared to WT, indicating a slight reduction of ubiquitination activity for the Asn4075Lys variant compared to WT. Further, a co-immunoprecipitation of the WT DUF domains and the Arg110Gln variant in HEK 293 cells were performed to investigate possible protein interaction partners binding to the DUF908/DUF913 domains of HUWE1. By quantitative mass spectrometry, twelve proteins (including HUWE1) were found to be significantly enriched/deprived in the co-immunoprecipitate compared to the negative control and the majority of those were overexpressed in WT DUF versus the Arg110Gln variant, but only three were significant. Further, 7/8 of these were identified as ribonucleic acid (RNA) binding and/or poly(A) RNA binding proteins by two different bioinformatics software programs. Especially interesting was the prolyl 4-hydroxylase beta (P4HB) protein as it is linked to Cole-Carpenters Syndrome 1, which shares a similar phenotype of craniosynostosis as the patients carrying the Arg110Gln variant. P4HB is a multifunctional protein that catalyzes formation, breakage and rearrangement of disulfide bonds, as well as functioning as a chaperone in higher concentrations. Another interesting candidate protein was FBXO22, another E3 ubiquitin ligase, which similar to HUWE1, can bind and ubiquitinate p53. To conclude, important tools to functionally study HUWE1 have been developed in this project. The DUF domains seem to interact with several proteins, where P4HB may be the most interesting candidate for future investigations, given its association with Cole-carpenter syndrome and craniosynostosis. Further, the Asn4075Lys appears to have some reduction in ubiquitination activity that could indicate a reduced activity mechanism for this variant.

Helena Amalie Hushagen, Kari Fladmark, Amanda Edson

Impact of cysteine 106 oxidation on function and localisation of the Parkinson’s disease-related protein DJ-1

DJ-1 is a multifunctional ubiquitously expressed protein encoded by the PARK7 gene in humans. Dysfunction of DJ-1 is implicated in various human diseases, particularly neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). The highly conserved cysteine residue at position 106 has been shown to be an important site for posttranslational modifications modulating the activity and localization of DJ-1, and acting as a sensor of oxidative and nitrosative stress in the cell. The potential neuroprotective role of astroglial DJ-1 as a regulator of antioxidant gene expression and as an antioxidant itself has been demonstrated in vitro and in multiple animal models exposed to oxidative stressors. To further our understanding of DJ-1’s protective role in astroglia and the importance of the conserved C106, we developed a mutant zebrafish line with astroglia-restricted expression of DJ-1C106A driven by the promotor region of the glial fibrillary acidic protein (GFAP) (park7-/-,Tg(gfap:egfp-2A-flag-zDJ-1C106A)) (UiB2003). The response of UiB2003 larvae to the PD phenotype-inducing mitochondrial complex I inhibitor rotenone was compared to that of wild type, DJ-1 knockout (DJ-1 KO) and a corresponding astroglial-restricted DJ-1WT-expressing zebrafish line (park7-/-,Tg(gfap:egfp-2A-flag-zDJ-1)) (UiB2001). Both UiB2003 and UiB2001 larvae displayed a drastically lower survival than WT and DJ-1 KO, as well as lowered heart rate compared to controls. The UiB2003 larvae also showed significantly higher incidence of edema than DJ-1 KOs. The expression of tyrosine hydroxylase (TH), a marker for dopaminergic cell death, was reduced in UiB2003 larvae compared to WT under basal conditions. The line did however sow a lower induction of inducible nitric oxide synthase (iNOS), a marker for intracellular stress, than DJ-1 KO larvae where marker was significantly increased. Adult UiB2003 fish showed a reduced complex I activity in skeletal muscle compared to WT fish, while the activity in the astroglia-rich brain was unaffected, indicating C106 independent effect of DJ-1 on complex I regulation.In order to examine the role of cysteine 106 on the ultrastructural localization of DJ-1 in response to oxidative stress, fusion proteins of DJ-1WT/DJ-1C106A and the peroxidase APEX2 were used to facilitate the targeted diaminobenzidine-staining of the SH-SY5Y neuroblastoma cells following treatment with rotenone. This produced a DJ-1 specific staining pattern that could be visualized by transmission electron microscopy and revealed a mainly cytosolic expression of DJ-1, with no staining in the mitochondrial interior, possibly with some faint expression at the outer mitochondrial membrane. Additionally, the overexpression of the DJ-1WT/DJ-1 induced increased contact between the endoplasmic reticulum (ER) and mitochondria compared to non-transfected cells. Both the localization pattern and induced ER-mitochondria contact was independent of the C106 and of rotenone-induced oxidative stress.
In conclusion, the astroglia-restricted expression of DJ-1C106A, does not protect zebrafish larvae from rotenone-induced oxidative insult and even displays a negative effect under baseline conditions. On the contrary, it appears to facilitate a stronger negative response and heightened sensitivity to the toxicant. On the other hand, DJ-1C106A retains its ability to regulate complex I in adult fish and iNOS in larvae. The C106 mutation does not affect the intracellular localization of DJ-1 nor the ability of DJ-1 overexpression to induce ER-mitochondria association.

Henrik Bendiksen, Sigrid S. Skånland (UiO)

Analysis of in vitro and in vivo drug responses in chronic lymphocytic leukemia

Jorunn Hjaltadottir, Lise B. Gundersen, Ingvild Aukrust

Characterization of HNF-4A mutants (p.R76W and p.R76Q) causing MODY1 and Fanconi syndrome

Maturity Onset Diabetes of the Young (MODY) is a rare form of (monogenic) diabetes, which is characterized by reduced insulin secretion due to pancreatic β-cell dysfunction. Two point mutations (p.R76W and p.R76Q) in the DBD domain of the gene encoding the Hepatocyte nuclear factor 4- alpha (HNF-4A) transcription factor have been associated with a patient phenotype of neonatal hyperinsulinaemic hypoglycaemia (HH) progressing into MODY1 in adult life, in addition to Fanconi syndrome. How these mutations affect the role of HNF-4A in the normal regulation of genes in target tissues like the liver, pancreas and kidney, remains unknown.The effect of the mutations on HNF-4A DNA binding, transactivation, and protein level were investigated, as well as on global gene expression and protein interaction in target cell lines. Both mutants (p.R76W and p.R76Q) showed a reduced DNA binding affinity for the key HNF-4A target promoters HNF1A and G6PC by EMSA analysis. Using a luciferase-linked reporter assay, a reduced transactivation activity for the HNF1A and G6PC promoter was detected for mutants in HepG2 and MIN6 cells, except for p.R76W in HepG2 cells, which showed an increased transactivation activity for the G6PC promoter.To see if the p.R76W mutation could alter recruitment of transcriptional coactivators of HNF-4A in a HepG2 cell system, mass spectrometry was performed on immunoprecipitated cell lysate to identify HNF-4A binding partners. Of 16000 identified proteins (1 % FDR), the only previously known HNF-4A coactivator CREB-binding protein was identified, however with similar abundance in WT versus the p.R76W sample. Furthermore, three proteins that are involved in the general transcriptional complex (PHD finger protein 6, Activated RNA polymerase II transcriptional coactivator p15 and FACT complex subunit SSRP1) where found to have a significantly lower abundance in the p.R76W sample compared to the WT sample. By mRNA sequencing of transfected HepG2 cells, the p.R76W mutant induced a significantly altered gene expression (both up- and downregulation) of genes involved in glucose and lipid metabolism. Of particular interest was the upregulation of the G6PC (1.7 fold change (log 2)) gene by the p.R76W mutant. An upregulation of HNF4A levels in the p.R76W sample, as well as an increased level of this mutant by immunoblot analyses of total cell (protein) lysate could suggest more stable mutant mRNA and protein. The p.R76W induced upregulation of G6PC and increased transactivation activity for the G6PC promoter in HepG2 cells is an interesting observation in relation to the unique phenotype of individuals with this HNF4A mutation (p.R76W). However further work is needed to confirm these findings and to link the genotype with the phenotype.

Marte Heimli, Anette Wolff, Øyvind Bruserud og Bergithe Oftedal

Characterization of regulatory T cells inAutoimmune Polyendocrine Syndrome type I, a model disease for autoimmunity

The T and B lymphocytes of the adaptive immune system face the challenge of correctly recognizing antigens originating from a vast number of rapidly evolving pathogens, while ignoring those pertaining to the host itself. A failure to ensure such self-tolerance may lead to autoimmune responses, the cause of a wide range of adverse pathologies. One self-tolerance mechanism is the negative selection of developing T cell progenitors with self-reactive capabilities in the thymus, another is the peripheral tolerance inferred by regulatory T cells (Tregs). The Autoimmune Regulator (AIRE), which induces the ectopic expression of tissuerestricted antigens in the thymus, has been indicated to be of importance in both central and peripheral tolerance. Mutations in the AIRE gene is the cause of autoimmune polyendocrine syndrome type I (APS-1), an established model disease for autoimmunity.
In this project, we aimed to characterize expression patterns in Tregs in APS-1, both at the protein and RNA levels. By assessment of the expression of pre-selected candidate genes, a number of subtle trends could be observed. It was indicated that the thymic output of Tregs was lower in APS-1 patients compared to healthy controls, but that the patient’s Tregs were capable of inducing expression of genes associated with an activated state. However, the observations failed to reach statistical significance, preventing reliable conclusions. The exception is a significant increase in the expression of mTOR, a downstream mediator of signaling though PKB/Akt. This is of interest as the inhibition of PKB/Akt by the PTEN phosphatase appear to be important for Treg lineage identity.
When using RNA sequencing to perform a global search for gene expression perturbations, a number of genes were found to exhibit significant changes in expression levels in Tregs from APS-1 patients compared to the controls. Among the genes indicated to be significantly perturbed are SKI and NOTCH-1, potentially affecting TGF-b signaling. TGF-b has been suggested to have implications in Treg suppressive function and in the induction of the regulatory T cell phenotype.
Further validation of these results, and elucidation of their functional implications, would be relevant for future work. Our hope is that continued work on Treg biology, and identification of clinically relevant biomarkers, can contribute to the successful implementation of this cell population in diagnostics and treatment of patients with immune-mediated diseases.