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.     

Development and Implementation of a High-Throughput Compound Screening Assay for Targeting Disrupted ER Calcium Homeostasis in Alzheimer's Disease

Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer''s disease (AD) pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER). Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.     

Treatment with the PARP inhibitor,niraparib, sensitizes colorectal cancer cell lines to irinotecan regardless of MSI/MSS status

Background

Cells with homologous recombination (HR) deficiency, most notably caused by mutations in the BRCA1 or BRCA2 genes, are sensitive to PARP inhibition. Microsatellite instability (MSI) accounts for 10-15% of colorectal cancer (CRC) and is hypothesized to lead to HR defects due to altered expression of Mre11, a protein required for double strand break (DSB) repair. Indeed, others have reported that PARP inhibition is efficacious in MSI CRC.

Methods

Here we examine the response to niraparib, a potent PARP-1/PARP-2 inhibitor currently under clinical evaluation, in MSI versus microsatellite stable (MSS) CRC cell lines in vitro and in vivo. We compiled a large panel of MSI and MSS CRC cell lines and evaluated the anti-proliferative activity of niraparib. In addition to testing single agent cytotoxic activity of niraparib, we also tested irinotecan (or SN-38, the active metabolite of irinotecan) activity alone and in combination with niraparib in vitro and in vivo.

Results

In contrast to earlier reports, MSI CRC cell lines were not more sensitive to niraparib than MSS CRC cell lines¸ suggesting that the MSI phenotype does not sensitize CRC cell lines to PARP inhibition. Moreover, even the most sensitive MSI cell lines had niraparib EC50s greater than 10 fold higher than BRCA-deficient cell lines. However, MSI lines were more sensitive to SN-38 than MSS lines, consistent with previous findings. We have also demonstrated that combination of niraparib and irinotecan was more efficacious than either agent alone in both MSI and MSS cell lines both in vitro and in vivo, and that niraparib potentiates the effect of irinotecan regardless of MSI status.

Conclusions

Our results support the clinical evaluation of this combination in all CRC patients, regardless of MSI status.     

Ca2+-dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells

The calcyclin-binding protein (CacyBP) binds calcyclin (S100A6) at physiological levels of [Ca(2+)] and is highly expressed in brain neurons. Subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca(2+)](i). Immunostaining indicates that CacyBP is present in the cytoplasm of unstimulated cultured neurons in which resting [Ca(2+)](i) is known to be approximately 50 nm. When [Ca(2+)](i) was increased to above 300 nm by KCl treatment, the immunostaining was mainly apparent as a ring around the nucleus. Such perinuclear localization of CacyBP was observed in untreated neuroblastoma NB-2a cells in which [Ca(2+)](i) is approximately 120 nm. An additional increase in [Ca(2+)](i) to above 300 nm by thapsigargin treatment did not change CacyBP localization. However, when [Ca(2+)](i) in NB-2a cells dropped to 70 nm, because of BAPTA/AM treatment, perinuclear localization was diminished. Ca(2+)-induced translocation of CacyBP was confirmed by immunogold electron microscopy and by fluorescence of NB-2a cells transfected with an EGFP-CacyBP vector. Recombinant CacyBP can be phosphorylated by protein kinase C in vitro. In untreated neuroblastoma NB-2a cells, CacyBP is phosphorylated on a serine residue(s), but exists in the dephosphorylated form in BAPTA/AM-treated cells. Thus, phosphorylation of CacyBP occurs in the same [Ca(2+)](i) range that leads to its perinuclear translocation.     

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

Here, two temperature sensitive promoters, P2 and P7, isolated from Bacillus subtilis, were characterized. The production of beta-galactosidase driven by these promoters was much higher at 45 degrees C than that at 37 degrees C both in Escherichia coli and B. subtilis and that the P2 promoter showed higher expression strength in B. subtilis at 45 degrees C. Thereby, an efficient temperature-inducible expression system was constructed by using P2 promoter in B. subtilis. Thus, we isolated and characterized a newly temperature inducible promoter and exploited it as a potential expression element in B. subtilis.     

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method ¹ to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.Download video file.^{(34M, mov)}