DEAF1 is a transcription factor linked to suicide and depression and is recurrently mutated in non-syndromic intellectual disorder (ID). In humans with major depressive disorder (MDD), DEAF1 is reported to have altered expression in the prefrontal cortex and the dorsal raphe nucleus of females but not males, and therefore may function in sex-specific depression. Standard (whole body) knockout of Deaf1 has been reported to alter 5-hydroxytryptamine (serotonin) receptor 1A (Htr1a) levels in the frontal cortex and dorsal raphe nucleus. We hypothesized that mice with targeted deletion of Deaf1 in brain would produce behavior phenotypes analogous to human MDD and ID, and that changes in Deaf1-target gene expression would be associated with behavior changes. To test this hypothesis, we produced a mouse line to allow conditional gene targeting of exons 2-5 (Deaf1-flox). Deaf1-flox mice were bred to congenic status onto a C57BL/6 background, and were then bred to mice transgenic for the Nestin-Cre gene to produce embryonic knockout of Deaf1 in neuronal precursor cells. Adult mice were tested for anxiety behavior using the Elevated Plus Maze and Open Field Exploration tests, and were tested for depression-like behavior using the Porsolt forced swim and sucrose preference tests. Relative to control mice, both male and female mice with homozygous deletion of Deaf1 in brain displayed increased anxiety measured by the anxiety tests, and no differences were measured in tests for depression-like behavior. Rotarod testing showed no deficits in motor skills of male mice. Learning and memory in mice were tested using Morris Water Maze (MWM) and fear conditioning. No change in long-term memory in male mice was observed in MWM, but both male and female mice lacking Deaf1 displayed severe deficits in fear conditioning tests. Eif4g3 and Tmem80 are target genes of Deaf1 and decreased mRNA levels were observed for Eif4g3 and Tmem80 throughout the brain of Deaf1 knockout mice, with no change in Htr1a mRNA levels. Our results demonstrate that conditional knockout of Deaf1 in neuronal precursors produces anxiety behavior and deficits in learning and memory in adult mice, potentially without changes in Htr1a mRNA levels, and that this mouse model may be useful in understanding the molecular mechanisms underlying MDD and ID in humans.

Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...

Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...