Functional and structural characterization of the slitrk2

ABSTRACT Background: Leucine-Rich Repeat (LRR) proteins play a key role in several systems, from immune response to neuritogenesis. Some years ago, our laboratory identified a gene located in the q27 region of the human X Chromosome that encodes for a LRR-enriched protein. Interestingly, this cytogenetic band contains the gene responsible for the Fragile X syndrome, the most common form of inherited intellectual disability, and other several tissue-specific genes, in particular several brain-specific genes. The function of the gene identified in our laboratory, located to on the q27.3 region and named Slitrk2, is to date largely unknown. The goal of this work was to characterize structurally and functionally Slitrk2. Principal Findings: Slitrk2 is a member of the Slitrk family. Slitrk family has 6 members whose function is still unknown. In order to achieve a better understanding about Slitrk2 function, we carried out integrated analysis at the transcriptomic, proteomic and bioinformatic level. At a transcriptomic level, we found that Slitrk2 expression is mainly restricted to the Central Nervous System. Slitrk2 is also expressed, although at low level compared to brain, in mammary gland, pancreas, prostate, testis. This expression pattern suggest a role for Slitrk2 not only in brain, but in general in organogenesis and morphogenesis. Slitrk2 expression during mouse embryogenesis starts at Embryonic Day 11, indicating that Slitrk2 acts in post-gastrulation mechanisms. By in situ hybridization in adult mouse brain, we found that Slitrk2 is expressed mainly in the Dentate Gyrus of the hippocampal formation. The in silico analysis of the protein reveals that Slitrk2 is likely to be located at the plasma-membrane. Some Trans-Golgi retrivial signals were found in the Slitrk2 sequence. With a multilevel computer-assisted approach we identified two potential phosphorylation sites: Threonine 806 and Tyrosine 832. Tyrosine 832 is in an aminoacidic environment able to be recognised by the COP-I coatomer, indicating that a Golgi-Retrivial of Slitrk2 is possible. The three-dimensional structure of Slitrk2 obtained with Phyre and I-TASSER calculations showed that the predicted extracellular region of Slitrk2 folded similarly to the ectodomain of other key neuronal protein such as Lingo-1 and NogoReceptor. With in vitro analyses, we found that Slitrk2 protein is mainly located intracellularly, being polarized to one side of the perinuclear boundary in PC12 cells. Slitrk2 expression is regulated by Nerve Growth Factor during PC12 cells differentiation, being downregulated after NGF exposure. The forced expression of Slitrk2 during NGF differentiation of PC12 cells lead to a reduction of the neuritic network caused by an impairment in Intermediate Filaments assembly (Peripherin). During NGF deprivation of neurotrophin-differentiated PC12 cells Slitrk2 transcript is upregulated. The knocking-down of Slitrk2 with short-interfering RNA recovered the drop in cell viability caused by neurotrophin depletion in NGF-differentiated PC12 cells. Outside the nervous system, Slitrk2 is expressed at low levels also in mammary gland. Also in mammary cell lines, both normal and tumoral (MCF10A and LA7/3F12, respectively), Slitrk2 forced expression determined impairment in differentiation reducing tubulogenesis. In both the above-mentioned mammary cell lines, immunofluorescence analysis showed that Slitrk2 localization is mainly intracellularly as seen in PC12 cells. Significance: In this work we performed: i) the transcriptional analysis of Slitrk2 in neuronal and non-neuronal systems; ii) the bioinformatic analysis of Slitrk2 topology, phosphorylations and three-dimensional structure and iii) the functional analysis of Slitrk2 in both neuronal and mammary gland cells. The main findings of this work are related to: i) the in vivo expression of Slitrk2 in adult mouse brain; ii) the homology modelling of the three-dimensional structure of Slitrk2 with the mapping of the predicted potential phosphorylation sites, potential N-glycosylation site and predicted ligand-binding sites and iii) the involment of Slitrk2 in the regulation of Intermediate Filament assembly in neuronal cells and the involment of Slitrk2 as a regulator of tubulogenesis in mammary cells. Future directions: In order to complete this work, we are currently analysing the role of Slitrk2 in embryosgenesis by using Danio rerio (Zebrafish) as a model of development. We are also studying more in deep the pathways by which Slitrk2 regulates neuronal cells differentiation. As last, we are now studying at the molecular level the role of Slitrk2 in tubulogenesis not only in mammary gland but also in kidney.