Molecular Mechanisms of Secreting Vesicle Biogenesis and Secretion in Chronic Degenerative Diseases

Regulated trafficking and secretion of insulin by the β cell of the endocrine pancreas is critical to maintain our body energy homeostasis. Disruption of these processes typically leads to hyperglycemia and the complications of diabetes. Compared to methods using anti-insulin or C-peptide antibodies, the fluorescent protein labeling approaches provide many advantages in live-cell, real time format with dynamic spatial and temporal monitoring. Previous studies from our lab demonstrated that by fusing a GFP within the C peptide of mouse proinsulin (Ins-C-GFP) insulin secretory granule targeting, trafficking and exocytosis could be monitored in live cells. Confocal microscopy and western blot results showed over 85% of the Ins-C-GFP can be targeted to insulin granules, with highly efficient proteolytic processing to mature insulin and C-GFP. Our present project aims to establish the minimum molecular determinants within human proinsulin required for its targeting to secretory granules. In order to do this, we designed a viral shuttle plasmid containing only the signal peptide, the first 5 residues of the B chain, followed by a monomeric GFP(B5), chemically synthesized with restriction sites for highly efficient and systematic chimeric and point mutagenesis. Confocal microscopy and 3-D reconstruction experiments revealed that the B5 vector was successfully expressed and nearly all of the fluorescent protein appeared within the ER(5 transfections; 72 cells), whereas the full-length hIns-C-emGFP vector efficiently targets insulin secretory granules. The results make it unlikely that the first five residues of the B chain are sufficient for human proinsulin targeting to secretory granules. The results also suggest that the middle of the C peptide is not necessary for human proinsulin targeting. We are presently characterizing a construct with the signal peptide alone without any insulin B chain residues (B0). By systematically adding back segments from hIns-C-emGFP to B5 or B0 and the following systematic point mutagenesis, we aim to establish the minimal segments and the precise residue(s) or motif(s) of human proinsulin required for targeting to secretory granules

Regulation of KLF4 Turnover Reveals an Unexpected Tissue-Specific Role of pVHL in Tumorigenesis

The transcription factor Krüppel-like factor 4 (KLF4) is an important regulator of cell fate decision, including cell cycle regulation, apoptosis, and stem cell renewal, and plays an ambivalent role in tumorigenesis as a tissue specific tumor suppressor or oncogene. Here we report that the Von Hippel-Lindau gene product, pVHL, physically interacts with KLF4 and regulates its rapid turnover observed in both differentiated and stem cells. We provide mechanistic insights into KLF4 degradation and show that pVHL depletion in colorectal cancer cells leads to cell cycle arrest concomitant with increased transcription of the KLF4-dependent p21 gene. Finally, immunohistochemical staining revealed elevated pVHL and reduced KLF4 levels in colon cancer tissues. We therefore propose that unexpectedly pVHL, via the degradation of KLF4, is a facilitating factor in colorectal tumorigenesis

Regulation of KLF4 Turnover Reveals an Unexpected Tissue-Specific Role of pVHL in Tumorigenesis

The transcription factor Krüppel-like factor 4 (KLF4) is an important regulator of cell fate decision, including cell cycle regulation, apoptosis, and stem cell renewal, and plays an ambivalent role in tumorigenesis as a tissue specific tumor suppressor or oncogene. Here we report that the Von Hippel-Lindau gene product, pVHL, physically interacts with KLF4 and regulates its rapid turnover observed in both differentiated and stem cells. We provide mechanistic insights into KLF4 degradation and show that pVHL depletion in colorectal cancer cells leads to cell cycle arrest concomitant with increased transcription of the KLF4-dependent p21 gene. Finally, immunohistochemical staining revealed elevated pVHL and reduced KLF4 levels in colon cancer tissues. We therefore propose that unexpectedly pVHL, via the degradation of KLF4, is a facilitating factor in colorectal tumorigenesis

APC/C-Cdh1: From cell cycle to cellular differentiation and genomic integrity

Anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin-protein ligase that targets various substrates for proteolysis inside and outside of the cell cycle. The activation of APC/C is dependent on two WD-40 domain proteins, Cdc20 and Cdh1. While APC/Cdc20 principally regulates mitotic progression, APC/Cdh1 shows a broad spectrum of substrates in and beyond cell cycle. In the past several years, numerous biochemical and mouse genetic studies have greatly attracted our attention to the emerging role of APC/Cdh1 in genomic integrity, cellular differentiation and human diseases. This review will aim to summarize the recently expanded understanding of APC/Cdh1 in regulating biological function and how its dysfunction may lead to diseases