An Alternatively Spliced Bifunctional Localization Signal Reprograms Human Shugoshin 1 to Protect Centrosomal Instead of Centromeric Cohesin

Separation of human sister chromatids involves the removal of DNA embracing cohesin ring complexes. Ring opening occurs by prophase-pathway-dependent phosphorylation and separase-mediated cleavage, with the former being antagonized at centromeres by Sgo1-dependent PP2A recruitment. Intriguingly, prophase pathway signaling and separase’s proteolytic activity also bring about centriole disengagement, whereas Sgo1 is again counteracting this licensing step of later centrosome duplication. Here, we demonstrate that alternative splice variants of human Sgo1 specifically and exclusively localize and function either at centromeres or centrosomes. A small C-terminal peptide encoded by exon 9 of SGO1 (CTS for centrosomal targeting signal of human Sgo1) is necessary and sufficient to drive centrosomal localization and simultaneously abrogate centromeric association of corresponding Sgo1 isoforms. Cohesin is shown to be a target of the prophase pathway at centrosomes and protected by Sgo1-PP2A. Accordingly, premature centriole disengagement in response to Sgo1 depletion is suppressed by blocking ring opening of an engineered cohesin

Identification of K_Ca3.1 Channel as a Novel Regulator of Oxidative Phosphorylation in a Subset of Pancreatic Carcinoma Cell Lines

<div><p>Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in <i>KRAS</i> as well as loss-of-function mutations in <i>p53</i>. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the <i>KCNN4</i> gene, which encodes for the Ca²⁺-sensitive K⁺ channel K_Ca3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to K_Ca3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of K_Ca3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC.</p></div

Optimization of screening conditions in Mia PaCa-2 cells.

<p><b>(A)</b> Oxygen consumption measurements (OCR) of Mia PaCa-2 cells in 25mM glucose or 25mM galactose. The Complex I inhibitor rotenone (1 μM) was used as a positive control for the inhibition of OxPhos, n = 6; <b>(B)</b> Measurement of OCR in media supplied with different carbon sources: 25 mM Glucose, 2 mM Glutamine, 2 mM Na Pyruvate, n = 12; <b>(C)</b> CellTiter Blue fluorescence measurements of cell titrations cultured in different assay media for 2 hrs, n = 12; <b>(D)</b> OCR measurements of Mia PaCa-2 cell titrations in different assay media, n = 12; <b>(E)</b> Nuclei number count using Hoechst stain in Seahorse plate, n = 12. For A, t-test was performed, *, p-value is 0.01 to 0.05; **, p-value is 0.001 to 0.01; B, Tukey’s Multiple Comparison ANOVA Test was performed. Groups are significantly different unless indicated otherwise. For C and D, two-tailed t-test was performed, ns, not significant, p>0.05, *, p-value is 0.01 to 0.05; **, p-value is < 0.01.</p

Metabolism of PDAC cell lines.

<p><b>(A)</b> Measurement of OCR, <b>(B)</b> ECAR, <b>(C)</b> OCR/ECAR and <b>(D)</b> metabolic phenotyping were performed in Seahorse assay medium supplied with 11 mM Glucose, 2 mM Pyruvate and 2 mM Glutamine. 20 000 cells/well were seeded one day prior to the experiment. n = 12 for each cell line. Tukey’s Multiple Comparison ANOVA test was performed. All groups are significantly different unless indicated otherwise.</p

K_Ca3.1 inhibition modulates oxygen consumption profile in a subset of PDAC cell lines.

<p><b>(A)</b> Seahorse XF Mito Stress test was performed to measure mitochondrial function upon various concentrations of rac-16 treatment of Mia PaCa-2, <b>(B)</b>, Panc-1, <b>(C)</b> BxPC-3, and <b>(D)</b> Capan-1 cells. n = 3. Oligo is oligomycin A, FCCP is carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, A/R is a mix of antimycin A and rotenone. n = 6.</p

Transportome hits which downregulate oxygen consumption >10%.

<p>Transportome hits which downregulate oxygen consumption >10%.</p

Transportome hits which upregulate oxygen consumption >10%.

<p>Transportome hits which upregulate oxygen consumption >10%.</p