BP1, a homeodomain-containing isoform of DLX4, represses the beta-globin gene.

In earlier studies we identified a putative repressor of the human beta-globin gene, termed beta protein 1 (BP1), which binds to two silencer DNA sequences upstream of the adult human beta-globin gene and to a negative control region upstream of the adult delta-globin gene. Further studies demonstrated an inverse correlation between the binding affinity of the BP1 protein for the distal beta-globin silencer sequence and the severity of sickle cell anemia, suggesting a possible role for BP1 in determining the production of hemoglobin S. We have now cloned a cDNA expressing the BP1 protein. Sequencing revealed that BP1 is a member of the homeobox gene family and belongs to the subfamily called Distal-less (DLX), genes important in early development. Further analysis showed that BP1 is an isoform of DLX4. BP1 protein has repressor function towards the beta-globin promoter, acting through the two beta-globin DNA silencers, demonstrated in transient transfection assays. Strong BP1 expression is restricted to placenta and kidney tissue, with no expression in 48 other human tissues. BP1 exhibits regulated expression in the human erythroid cell line MB-02, where its expression decreases upon induction of the beta-globin gene. BP1 is thus the first member of the DLX family with known DNA binding sites and a function in globin gene regulation.

Ubiquilin regulates presenilin endoproteolysis and modulates gamma-secretase components, Pen-2 and nicastrin.

Mutations in presenilin proteins (PS1 and PS2) lead to early-onset Alzheimer's disease. PS proteins are endoproteolytically cleaved into two main fragments: the NTF (PS N-terminal fragment) and the CTF (PS C-terminal fragment). The two fragments are believed to constitute the core catalytic enzyme activity called gamma-secretase, which is responsible for cleaving beta-amyloid precursor protein to release Abeta. Thus, studying factors that modulate PS fragment levels could provide important information about gamma-secretase. Previously, we demonstrated that the protein, ubiquilin-1, interacts both in vivo and in vitro with PS and that overexpression of ubiquilin-1 or -2 leads to increased accumulation of full-length PS proteins. Using wild-type HEK-293 cells (human embryonic kidney 293 cells) and PS-inducible cells, we now show that overexpression of either ubiquilin-1 or -2 decreases the PS NTF and CTF levels. Conversely, siRNA (small interfering RNA)-mediated knockdown of ubiquilin-1 and -2 proteins increased the PS NTF and CTF levels. We considered that ubiquilin might alter PS fragment accumulation by acting as a shuttle factor escorting PS fragments to the proteasome for degradation. However, through proteasome inhibition studies, we show that this does not occur. Instead, our results suggest that ubiquilin regulates PS fragment production. We also examined whether other components of the gamma-secretase complex are affected by ubiquilin expression. Interestingly, overexpression of ubiquilin resulted in a decrease in Pen-2 and nicastrin levels, two essential components of the gamma-secretase complex. In contrast, knockdown of ubiquilin-1 and -2 protein expression by RNAi (RNA interference) increased Pen-2 and nicastrin levels. Finally, we show that inhibition of the proteasome results in decreased PS fragment production and that reversal of proteasome inhibition restores PS fragment production, suggesting that the proteasome may be involved in PS endoproteolysis. These studies implicate ubiquilin as an important factor in regulating PS biogenesis and metabolism.

Overexpression of ubiquilin decreases ubiquitination and degradation of presenilin proteins.

Mutations in presenilin proteins (PS1 and PS2) are associated with most cases of early-onset Alzheimer's disease. Several proteins appear to regulate accumulation of PS proteins in cells. One such protein is ubiquilin-1, which increases levels of coexpressed PS2 protein in a dose-dependent manner. We now report that overexpression of ubiquilin-2, which is 80% identical to ubiquilin-1, also increases the levels of coexpressed PS1 and PS2 proteins in cells. To investigate the mechanism by which ubiquilin proteins increase levels of PS proteins, we examined how overexpression of ubiquilin-1, which possesses all of the key signature motifs present in ubiquilin proteins, affects PS2 gene transcription and PS2 protein turnover and ubiquitination. HeLa cells overexpressing both PS2 and ubiquilin-1 had PS2 mRNA levels lower than HeLa cells overexpressing PS2 alone, indicating that ubiquilin-1 overexpression, in fact, decreases PS2 transcription. Cells overexpressing ubiquilin-1 and PS2 displayed decreased turnover of high molecular weight (HMwt) forms of PS2 but not of full-length PS2 proteins. The reduced turnover of HMwt PS2 proteins appears to be mediated by the binding of the ubiquitin-associated domain (UBA) of ubiquilin to ubiquitin chains conjugated onto PS2 proteins. Immunoprecipitation studies indicated that ubiquilin-1 overexpression decreases ubiquitination of coexpressed PS2 proteins, suggesting that binding of ubiquilin might block ubiquitin chain elongation. Consistent with this model, we found that the UBA domain of ubiquilin-1 binds poly-ubiquitin chains in vitro. In addition, we show that ubiquilin proteins colocalize with ubiquitin-immunoreactive structures in cells and that ubiquilin proteins are present within the inner core of aggresomes, which are structures associated with accumulation of misfolded proteins in cells. Our results suggest that ubiquilin proteins play an important role in regulating PS protein levels in cells.