Density of Sgt1-immunopositive neurons is decreased in the cerebral cortex of Alzheimer's disease brain.

Sgt1 was discovered as a protein required for the mitotic activity of kinetochore and for the activity of ubiquitin ligase in yeast [Kitagawa, K., Skowyra, D., Elledge, S.J., Harper, J.W., Hieter, P., 1999. SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex. Mol. Cell 4, 21-33.]. Later, Sgt1 was identified in different organisms including mammals where it was found at high level in the brain. To understand Sgt1 function in this tissue we analyzed its localization in human brain by immunohistochemistry. In normal brain we observed Sgt1-immunostaining in Purkinje cells of the cerebellum, in granule cells of the dentate gyrus of the hippocampus and in multiple neurons of the cortex. By Western blotting we found a higher level of this protein in the cortex than in the cerebellum. Subsequent morphometric analyses showed that the density of Sgt1-immunopositive neurons varied in different cortical regions. The highest density of Sgt1-immunopositive cells was seen in the temporal cortex (from 1.2% to 5.7%), and the lowest - in the entorhinal cortex (from 0 to 1.1% of all neurons). We next compared the density of Sgt1-immunopositive neurons in cortical layers of healthy aged and Alzheimer's disease (AD) brain sections. A significant decrease in Sgt1-immunopositive neurons was found in the temporal (up to 25-fold), angular (up to 11-fold) and posterior cingulate cortex (up to five-fold). In the entorhinal and precentral cortex the reduction of Sgt1-immunopositive neurons was only about two-fold in AD brains as compared to healthy aged ones. The presence of Sgt1 in post-mitotic neurons indicates the involvement of this protein in a process different from that required for activity of the kinetochore. Decreased immunostaining in AD cortex point to Sgt1 as a possible marker of neurons degenerating in AD.

The expression and function of Sgt1 protein in eukaryotic cells.

The Sgt1 protein was discovered in yeast but later it was found in other eucaryotic organisms, including plants and mammals. It was suggested that the Sgt1 protein plays a role in the CBF3 kinetochore and the SCF ubiquitin ligase complexes. Since the distribution, properties and functions of the mammalian Sgt1 have not been extensively studied, we examined the expression of Sgt1 protein in the rat tissues and here we report the preliminary results. Using northern and western blots we found that the Sgt1 protein is expressed in various tissues and that brain, skeletal muscles and spleen contain high levels of this protein and its mRNA. Then, by western blot, we checked the localization of Sgt1 in some areas of the rat brain such as cortex, cerebellum and hippocampus and we found that cerebellum and cortex contain highest amounts of this protein. Using immunohistochemistry we found that the Sgt1 protein is present in both neurons and glial cells.

Hsp70 is a new target of Sgt1--an interaction modulated by S100A6.

In this work, we identified Hsp70 as a novel target of the Sgt1 protein. Using co-immunoprecipitation, affinity chromatography and ELISA we showed that, besides Hsp90, Sgt1 interacts with the heat shock protein, Hsp70. We also found that a deletion mutant of Sgt1, devoid of the C-terminal region, did not bind to either Hsp70 or Hsp90 proteins. Overexpression of S100A6, a calcium binding protein that interacts with the C-terminal part of Sgt1, decreased the amount of chaperone bound to Sgt1. However, the effect of S100A6 on this interaction was not observed in BAPTA/AM treated cells in which Ca(2+) level was decreased. This suggests that the interaction of Sgt1 with Hsp70 and Hsp90 is regulated by S100A6 in a Ca(2+)-dependent manner.

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins.

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.