Identification of the Degradation Determinants of Insulin Receptor Substrate 1 for Signaling Cullin-RING E3 Ubiquitin Ligase 7-mediated Ubiquitination

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase S6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain PI3K activity and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser-307/Ser-312 and Ser-527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyperphosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1–260 led to substantial reduction of ubiquitination efficiency, suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and the N terminus of IRS1 for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.     

Inhibition of stearoyl-coA desaturase selectively eliminates tumorigenic Nanog-positive cells: Improving the safety of iPS cell transplantation to myocardium

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However, residual undifferentiated iPS derivates (iPSD), which are not fully eliminated by cell differentiation or purification protocols, may form tumors after transplantation, thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells, which share many features with iPSD. Here, we tested the effects of PluriSin#1, a small-molecule inhibitor of SCD, on iPS-derived CM. We found that plurisin#1 treatment significantly decreased the mRNA and protein level of Nanog, a marker for both cell pluripotency and tumor progression; importantly, we provide evidence that PluriSin#1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition, PluriSin#1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin#1 treatment prevents tumorigenicity of iPSD after cell transplantation, we intramyocardially injected PluriSin#1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection, which was prevented by treatment with PluriSin#1. Moreover, treatment with PluriSin#1 did not change the expression of cTnI, α-MHC, or MLC-2v, markers of cardiac differentiation (P > 0.05, n = 4). Importantly, pluriSin#1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration.     

Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.     

CCL7 contributes to angiotensin II-induced abdominal aortic aneurysm by promoting macrophage infiltration and pro-inflammatory phenotype

Chemokine C-C motif ligand 7 (CCL7), a member of CC chemokine subfamily, plays pivotal roles in numerous inflammatory diseases. Hyper-activation of inflammation is an important characteristic of abdominal aortic aneurysm (AAA). Therefore, in the present study, we aimed to determine the effect of CCL7 on AAA formation. CCL7 abundance in aortic tissue and macrophage infiltration were both increased in angiotensin II (Ang II)-induced AAA mice. Ex vivo, CCL7 promoted macrophage polarization towards M1 phenotype. This effect was reversed by the blockage of CCR1, a receptor of CCL7. CCL7 up-regulated JAK2/STAT1 protein level in macrophage, and CCL7-induced M1 activation was suppressed by JAK2/STAT1 pathway inhibition. To verify the effect of CCL7 on AAA in vivo, either CCL7-neutralizing antibody (CCL7-nAb) or vehicles were intraperitoneally injected 24 hours prior to Ang II infusion and subsequently every three days for 4 weeks. CCL7-nAb administration significantly attenuated Ang II-induced luminal and external dilation as well as pathological remodelling. Immunostaining showed that CCL7-nAb administration significantly decreased aneurysmal macrophage infiltration. In conclusion, CCL7 contributed to Ang II-induced AAA by promoting M1 phenotype of macrophage through CCR1/JAK2/STAT1 signalling pathway.     

Combined Strategy of Endothelial Cells Coating,Sertoli Cells Coculture and Infusion Improves Vascularization and Rejection Protection of Islet Graft

Improving islet graft revascularization and inhibiting rejection become crucial tasks for prolonging islet graft survival. Endothelial cells (ECs) are the basis of islet vascularization and Sertoli cells (SCs) have the talent to provide nutritional support and exert immunosuppressive effects. We construct a combined strategy of ECs coating in the presence of nutritious and immune factors supplied by SCs in a co-culture system to investigate the effect of vascularization and rejection inhibition for islet graft. In vivo, the combined strategy improved the survival and vascularization as well as inhibited lymphocytes and inflammatory cytokines. In vitro, we found the combinatorial strategy improved the function of islets and the effect of ECs-coating on islets. Combined strategy treated islets revealed higher levels of anti-apoptotic signal molecules (Bcl-2 and HSP-32), survival and function related molecules (PDX-1, Ki-67, ERK1/2 and Akt ) and demonstrated increased vascular endothelial growth factor receptor 2 (KDR) and angiogenesis signal molecules (FAk and PLC-γ). SCs effectively inhibited the activation of lymphocyte stimulated by islets and ECs. Predominantly immunosuppressive cytokines could be detected in culture supernatants of the SCs coculture group. These results suggest that ECs-coating and Sertoli cells co-culture or infusion synergistically enhance islet survival and function after transplantation.     

Inhibition Effect of a Custom Peptide on Lung Tumors

Cecropin B is a natural antimicrobial peptide and CB1a is a custom, engineered modification of it. In vitro, CB1a can kill lung cancer cells at concentrations that do not kill normal lung cells. Furthermore, in vitro, CB1a can disrupt cancer cells from adhering together to form tumor-like spheroids. Mice were xenografted with human lung cancer cells; CB1a could significantly inhibit the growth of tumors in this in vivo model. Docetaxel is a drug in present clinical use against lung cancers; it can have serious side effects because its toxicity is not sufficiently limited to cancer cells. In our studies in mice: CB1a is more toxic to cancer cells than docetaxel, but dramatically less toxic to healthy cells.     

From QTL to QTN: Candidate Gene Set Approach and a Case Study in Porcine IGF1-FoxO Pathway

Unraveling the genetic background of economic traits is a major goal in modern animal genetics and breeding. Both candidate gene analysis and QTL mapping have previously been used for identifying genes and chromosome regions related to studied traits. However, most of these studies may be limited in their ability to fully consider how multiple genetic factors may influence a particular phenotype of interest. If possible, taking advantage of the combined effect of multiple genetic factors is expected to be more powerful than analyzing single sites, as the joint action of multiple loci within a gene or across multiple genes acting in the same gene set will likely have a greater influence on phenotypic variation. Thus, we proposed a pipeline of gene set analysis that utilized information from multiple loci to improve statistical power. We assessed the performance of this approach by both simulated and a real IGF1-FoxO pathway data set. The results showed that our new method can identify the association between genetic variation and phenotypic variation with higher statistical power and unravel the mechanisms of complex traits in a point of gene set. Additionally, the proposed pipeline is flexible to be extended to model complex genetic structures that include the interactions between different gene sets and between gene sets and environments.