Biophysical understanding of amorphous protein aggregation can significantly impact diverse area of biotechnology. Here, we report the time dependent salt-induced formation of amorphous aggregation as monitored by fluorescence self-quenching and compare the results with conventional methods for detecting protein aggregation [static light scattering (LS) and dynamic light scattering (DLS)]. As a model protein, we used a bovine pancreatic trypsin inhibitor (BPTI) variant extended by two glycines (C2G) at its C terminus, and three variants where three types of Solubility Controlling Peptide tags (SCP tags) made of five serines (C5S), alanines (C5A) or aspartic acids (C5D) were added to the C terminus of C2G. All variants have a native-like BPTI structure and trypsin inhibitory activity, but different solubilities controlled by the SCP tags. The BPTIs were labeled using NHS-Fluorescein (FAM) conjugated to BPTI's lysines, and we measured the changes in fluorescence intensity occurring upon the addition of NaCl. The fluorescence of all FAM-BPTIs decreased almost immediately, albeit to a different extent, upon addition of salt and became constant after 10 min for 24 h or more. On the other hand, LS and DLS signal changes were dependent on the type of tags. Namely, C2G's LS and DLS signals changed immediately, the signals of C5S and C5A tagged FAM-BPTIs increased slowly from 10 min to 24 h, and those of C5D remained constant. These observations indicated the presence of at least one intermediate step, with increased protein-protein interaction yielding a 'molecular condensation' phase. According to this model, C2G would rapidly turn from 'condensates' to aggregates, whereas C5S and C5A tagged FAM-BPTIs would do so slowly, and the soluble C5D tagged variant would remain in the molecular condensation state.

Inflammatory bowel diseases (IBDs) are characterized by chronic inflammation involving intestinal tissue damage, which include ulcerative colitis and Crohn's disease as major entities. Accumulating evidence suggests that excessive apoptosis of intestinal epithelial cells (IECs) contributes to the development of IBD. It was recently reported that the transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) is involved in inflammation; however, its role in colitis remains unclear. Here, we found that C/EBPδ knockout mice showed enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis, a mouse model of IBD, which was associated with severe colonic inflammation and mucosal damage with increased IEC apoptosis.Additionally, DSS stimulation induced increased expression of pro-apoptotic BH3only protein Bim in the colon of C/EBPδ knockout mice. Collectively, our findings demonstrate that C/EBPδ plays an essential role in suppressing DSS-induced colitis, likely by attenuating IEC apoptosis.

<p>Loss of Dok-1/-2 enhances intestinal tumor growth in Apc mice.</p>

<p>mRNA expression of Dok-1 and Dok-2, but not Dok-3, is detectable in tumor epithelial cells.</p>

<p>Dok-3 deficiency causes malignant progression of colorectal tumors of Apc mice.</p>