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Cystatin C is a potent cysteine protease inhibitor that plays an important role in various biological processes including cancer, cardiovascular diseases and neurodegenerative diseases. However, the role of CstC in inflammation is still unclear. In this study we demonstrated that cystatin C-deficient mice were significantly more sensitive to the lethal LPS-induced sepsis. We further showed increased caspase-11 gene expression and enhanced processing of pro-inflammatory cytokines IL-1β and IL-18 in CstC KO bone marrow-derived macrophages (BMDM) upon LPS and ATP stimulation. Pre-treatment of BMDMs with the cysteine cathepsin inhibitor E-64d did not reverse the effect of CstC deficiency on IL-1β processing and secretion, suggesting that the increased cysteine cathepsin activity determined in CstC KO BMDMs is not essential for NLRP3 inflammasome activation. The CstC deficiency had no effect on (mitochondrial) reactive oxygen species (ROS) generation, the MAPK signaling pathway or the secretion of anti-inflammatory cytokine IL-10. However, CstC-deficient BMDMs showed dysfunctional autophagy, as autophagy induction via mTOR and AMPK signaling pathways was suppressed and accumulation of SQSTM1/p62 indicated a reduced autophagic flux. Collectively, our study demonstrates that the excessive inflammatory response to the LPS-induced sepsis in CstC KO mice is dependent on increased caspase-11 expression and impaired autophagy, but is not associated with increased cysteine cathepsin activity.

Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. Histone chaperones are regulators of this process by preventing formation of non-nucleosomal histone–DNA complexes. Aprataxin and polynucleotide kinase like factor (APLF) is a non-homologous end-joining (NHEJ) DNA repair factor that possesses histone chaperone activity in its acidic domain (APLFAD). Here, we studied the molecular basis of this activity using biochemical and structural methods. We find that APLFAD is intrinsically disordered and binds histone complexes (H3-H4)2 and H2A-H2B specifically and with high affinity. APLFAD prevents unspecific complex formation between H2A-H2B and DNA in a chaperone assay, establishing for the first time its specific histone chaperone function for H2A-H2B. On the basis of a series of nuclear magnetic resonance studies, supported by mutational analysis, we show that the APLFAD histone binding domain uses two aromatic side chains to anchor to the α1–α2 patches on both H2A and H2B, thereby covering most of their DNA-interaction surface. An additional binding site on both APLFAD and H2A-H2B may be involved in the handoff between APLF and DNA or other chaperones. Together, our data support the view that APLF provides not only a scaffold but also generic histone chaperone activity for the NHEJ-complex.

Triple naegative breast cancer has an increased rate of distant metastasis and consequently poor prognosis. To metastasize, breast cancer cells must detach from the main tumour mass and resist anoikis, a programmed cell death induced by lack of cell-extracellular matrix communication. Although cancer cells must detach to metastasize in vivo, the viability of floating cancer cells in vitro is rarely investigated. Here we show that co-treatment of anoikis-resistant MDA-MB-231 cells with metformin and 2-deoxy-D-glucose (2-DG) increased the percentage of floating cells, of which about 95% were viable. Floating cells resumed their proliferation once they were reseeded in the pharmacological compound-free medium. Similar effects on detachment were observed on anoikis-prone MCF-7 cells. Co-treatment of MDA-MB-231 cells with metformin and 2-DG induced a strong activation of AMP-activated protein kinase (AMPK), which was reduced by AMPK inhibitor compound C that prevented detachment of MDA-MB-231 cells. However, direct AMPK activators A-769662 and AICAR did not have any major effect on the percentage of floating MDA-MB-231 cells, indicating that AMPK activation is necessary but not sufficient for triggering detachment of cancer cells. Our results demonstrate that separate analysis of floating and attached cancer cells might be important for evaluation of anti-cancer agents.

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A2 (sPLA2) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA2. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b'. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA2s have the same fold as Atx. The first three sPLA2s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA2 in the Atx-hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA2s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA2 and hPDI is however different from that of the other sPLA2s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA2s confirmed the model-based expectation that GV sPLA2 was a more effective inhibitor than GX sPLA2, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA2s by assisting the retrograde transport of these molecules from the cell surface. The sPLA2-hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA2s in relation to their action intracellularly.