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Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions including cell cycle regulation and apoptosis. However, defining specific mechanisms and functions of sphingolipids in cell cycle regulation is missing. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes: The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, GIM4) as synthetically lethal with ISC1. The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1. Reciprocally, ISC1 deletion mutants were sensitive to Benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus, coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions. [ABSTRACT FROM AUTHOR]

Objectives:Fibrosis is characterized by excessive tissue remodeling resulting from altered expression of various growth factors, cytokines and proteases. We hypothesized that matrix metalloproteinase (MMP) mediated degradation of type IV collagen, a main component of the basement membrane, will release peptide fragments (neo-epitopes) into the circulation. Here we present the development of two competitive enzyme-linked immunosorbent assays (ELISAs) for assessing the levels of specific fragments of type IV collagen α1 (C4M12a1) and α3 (C4M12a3) chains in serum as indicators of fibrosis. Methods:Fragments of type IV collagen cleaved invitro by MMP-12 were identified by mass spectrometry, and two were chosen for ELISA development due to their unique sequences. The assays were evaluated using samples from a carbon tetrachloride (CCl4) rat model of liver fibrosis and from patients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD). Results:Two technically robust ELISAs were produced using neo-epitope specific monoclonal antibodies. Mean serum C4M12a1 levels were significantly elevated in CCl4-treated rats compared with controls in weeks 12, 16, and 20, with a maximum increase of 102% at week 16 (p < 0.0001). Further, C4M12a1 levels correlated with the total collagen content of the liver in CCl4-treated rats (r = 0.43, p = 0.003). Mean serum C4M12a3 levels were significantly elevated in patients with mild, moderate, and severe IPF, and COPD relative to healthy controls, with a maximum increase of 321% in COPD (p < 0.0001). Conclusions:Two assays measuring C4M12a1 and C4M12a3 enabled quantification of MMP mediated degradation of type IV collagen in serum. C4M12a1 was elevated in a pre-clinical model of liver fibrosis, and C4M12a3 was elevated in IPF and COPD patients. This suggests the use of these assays to investigate pathological remodeling of the basement membrane in different organs. However, validations in larger clinical settings are needed. [ABSTRACT FROM AUTHOR]