Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

Background: Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. Results: It was found that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. Conclusion: PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis

ERK1/2-Akt1 Crosstalk Regulates Arteriogenesis in Mice and Zebrafish

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis

Therapeutic Implications of GIPC1 Silencing in Cancer

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers

Fibroblast growth factor signaling potentiates VE-cadherin stability at adherens junctions by regulating SHP2.

The fibroblast growth factor (FGF) system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization.In vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs) known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity.These results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability

Identification of a Torpedo homolog of Sam68 that interacts with the synapse organizing protein rapsyn

AbstractNicotinic acetylcholine receptors (nAChRs) are initially expressed diffusely on the surface of myotubes and, in response to neuronally derived factors, cluster at the endplate to a final concentration of approximately 10 000/μm2. The synaptic peripheral membrane protein rapsyn has been shown to mediate clustering of nAChRs in several systems. Here we describe the use of the yeast two-hybrid system to identify proteins that can interact with rapsyn. One of the clones we have identified is a Torpedo californica homolog of the Src-associated in mitosis protein (Sam68). We further show that Sam68, like rapsyn, is localized at the neuromuscular junction

The redox-sensing protein Rex modulates ethanol production in <i>Thermoanaerobacterium saccharolyticum</i>

<div><p><i>Thermoanaerobacterium saccharolyticum</i> is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type <i>T</i>. <i>saccharolyticum</i>: a single deletion of <i>rex</i> resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the <i>rex</i> gene also had the effect of increasing expression of alcohol dehydrogenase genes, <i>adhE</i> and <i>adhA</i>. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δ<i>rex</i> strains adapted for faster growth. In several cases, secondary mutations had appeared in the <i>adhE</i> gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce <i>rex</i> into the Δ<i>rex</i> strains: reintroducing <i>rex</i> decreased ethanol yield to below wild type levels in the Δ<i>rex</i> strain without <i>adhE</i> mutations, but did not change the ethanol yield in the Δ<i>rex</i> strain where an <i>adhE</i> mutation occurred.</p></div

SHP2 protein stability is impaired in cells lacking FGF signaling.

<p><b>A.</b> SHP2 mRNA levels were not decreased in endothelial cells lacking FGF signaling. Quantitative RT-PCR analysis of total RNA isolated from BAEC. Total RNA was isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN. SHP2 mRNA levels were measured with real-time PCR and normalized to GAPDH expression (Mean ± SD, *<i>P</i><0.05, by t-test compared with NT). NT denotes no transduction. <b>B</b>. Western blotting of total cell lysates isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN and treated with 10 µg/ml cycloheximide for up to 36 hours. <b>C</b>. Quantitative analysis of SHP2 Western analysis described in B. The value at time point 0 was designated as 1. (n = 3 Mean ± SD, *<i>P</i><0.05, by t-test compared with Ad-GFP). <b>D</b>. SHP2 is degraded via the lysosomal pathway in the absence of FGF signaling. Confluent BAEC transduced with either Ad-GFP or Ad-FGFR1DN were treated with 1 µM MG132, 20 µM lactacystin, 20 µM chloroquine or 25 mM NH₄Cl for 24 hr. Total cell lysates were analyzed by Western blot. <b>E</b>. Quantitative analysis of SHP2 expression shown in Fig. 2D. The value of Ad-GFP control (DMSO) treatment, standardized with β-tubulin, was designated as 1. (n = 3, Mean ± SD, *<i>P</i><0.05, by t-test compared with Ad-FGFR1DN control).</p