Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10.

Two different mutated forms of BRI2 protein are linked with familial British and Danish dementias, which present neuropathological similarities with Alzheimer's disease. BRI2 is a type II transmembrane protein that is trafficked through the secretory pathway to the cell surface and is processed by furin and ADAM10 (a disintegrin and metalloproteinase domain 10) to release secreted fragments of unknown function. Its apparent molecular mass (42-44 kDa) is significantly higher than that predicted by the number and composition of amino acids (30 kDa) suggesting that BRI2 is glycosylated. In support, bioinformatics analysis indicated that BRI2 bears the consensus sequence Asn-Thr-Ser (residues 170-173) and could be N-glycosylated at Asn170. Given that N-glycosylation is considered essential for protein folding, processing and trafficking, we examined whether BRI2 is N-glycosylated. Treatment of HEK293 (human embryonic kidney) cells expressing BRI2 with the N-glycosylation inhibitor tunicamycin or mutation of Asn170 to alanine reduced its molecular mass by ~2 kDa. These data indicate that BRI2 is N-glycosylated at Asn170. To examine the effect of N-glycosylation on BRI2 trafficking at the cell surface, we performed biotinylation and (35)S methionine pulse-chase experiments. These experiments showed that mutation of Asn170 to alanine reduced BRI2 trafficking at the cell surface and its steady state levels at the plasma membrane. Furthermore, we obtained data indicating that this mutation did not affect cleavage of BRI2 by furin or ADAM10. Our results confirm the theoretical predictions that BRI2 is N-glycosylated at Asn170 and show that this post-translational modification is essential for its expression at the cell surface but not for its proteolytic processing.

Crosstalk mechanisms between the WNT signaling pathway and long non-coding RNAs.

The WNT/ß-catenin signaling pathway controls a plethora of biological processes throughout animal development and adult life. Because of its fundamental role during animal lifespan, the WNT pathway is subject to strict positive and negative multi-layered regulation, while its aberrant activity causes a wide range of pathologies, including cancer. At present, despite the inroads into the molecules involved in WNT-mediated transcriptional responses, the fine-tuning of WNT pathway activity and the totality of its target genes have not been fully elucidated. Over the past few years, long non-coding RNAs (lncRNAs), RNA transcripts longer that 200nt that do not code for proteins, have emerged as significant transcriptional regulators. Recent studies show that lncRNAs can modulate WNT pathway outcome by affecting gene expression through diversified mechanisms, from the transcriptional to post-translational level. In this review, we selectively discuss those lncRNA-mediated mechanisms we believe the most important to WNT pathway modulation.

A Positive Regulatory Loop between a Wnt-Regulated Non-coding RNA and ASCL2 Controls Intestinal Stem Cell Fate.

The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation, and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/ß-catenin transcriptional complex is the primary transforming factor in colorectal cancer. We identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/ß-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/ß-catenin to mediate the juxtaposition of its promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 transcriptionally, closing a feedforward regulatory loop that controls stem cell-related gene expression. This regulatory circuitry is highly amplified in colorectal cancer and correlates with increased metastatic potential and decreased patient survival. Our results uncover the interplay between non-coding RNA-mediated regulation and Wnt signaling and point to the diagnostic and therapeutic potential of WiNTRLINC1.

BRI2 interacts with BACE1 and regulates its cellular levels by promoting its degradation and reducing its mRNA levels.

BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid Precursor Protein (APP) and reduces the levels of secreted APPß (sAPPß), which derives from APP cleavage by ß-secretase (BACE1). Exploring the mechanisms of this effect, we obtained data that BRI2 decreases the cellular levels of BACE1 thus reducing the ß-cleavage of APP. Deletion of N-terminal cytoplasmic or C-terminal extracellular sequences of BRI2 neither affected its interaction with BACE1 or APP (Fotinopoulou et al., 2005) nor the reduction in the levels of BACE1 and sAPPß. These results suggest that BRI2 may prevent access of BACE1 to APP and the BRI2/BACE1 interaction may mediate the reduction in BACE1 levels. In support, BRI2 expression induced lysosomal but not proteasomal degradation of BACE1. In parallel, BRI2 expression was also found to reduce BACE1 mRNA levels by 50%. This study adds novel information regarding the mechanism by which BRI2 affects APP processing and BACE1 levels.