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2 The Pathway and Mechanism of Initiation of Protein Synthesis

T1 - Metabolic pathway structures for recombinant protein synthesis in Escherichia coli

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22/10/1999 · Mechanism of Protein Synthesis

N2 - Escherichia coli is a valuable commercial host for the production of heterologous proteins. We used elementary mode analysis to identify all possible genetically independent pathways for the production of three specific recombinant proteins, green fluorescent protein, savinase and an artificial protein consisting of repeating units of a five-amino-acid cassette. Analysis of these pathways led to the identification of the most efficient pathways for the production of each of these proteins. The results indicate that the amino acid composition of expressed proteins has a profound effect on the number and identity of possible pathways for the production of these proteins. We show that several groups of elementary modes produce the same ratio of biomass and recombinant protein. The pattern of occurrence of these modes is dependent on the amino acid composition of the specific foreign protein produced. These pathways are formed as systemic combinations of other pathways that produce biomass or foreign protein alone after the elimination of fluxes in specific internal reversible reactions or the reversible carbon dioxide exchange reaction. Since these modes represent pathway options that enable the cell to produce biomass and protein without utilizing these reactions, removal of these reactions would constrain the cells to utilize these modes for producing biomass and foreign protein at constant ratios.

a change in one protein in a biosynthetic pathway

Somatostatin is the natural neuropeptide produced in the pancreas by the δ islet cells. We have previously reported that somatostatin inhibits the PI3K/mTOR pathway and thereby protein translation at two levels: firstly, it directly inhibits PI3K activity (Bousquet et al, ; Najib et al, ), and secondly, it upregulates the expression of the hypophosphorylated form of the translation inhibitor 4E-BP1 (Azar et al, ; Laval et al, ). Somatostatin analogues (e.g. octreotide) have been FDA-approved and safely used for decades in the diagnosis (octreoscan) and treatment of secretory syndrome and growth of neuroendocrine tumours including pancreatic neuroendocrine tumours that highly express one or several of the five G protein-coupled somatostatin receptors (sst1, 2, 3 & 5) (Bousquet et al, ; Chalabi et al, ). However, octreotide, which chiefly targets sst2, has shown no clinical benefit for PDAC patients, which is probably explained by the absence of expression of sst2 in pancreatic cancer cells (Friess et al, ; Buscail et al, ; Laklai et al, ), or in CAFs, as demonstrated here (Supplementary Fig S5). Conversely, the new generation of somatostatin analogues including SOM230 which also targets the sst1 receptor subtype is shown here to be a very promising drug for the inhibition of CAF secretory activity, thereby re-sensitizing pancreatic cancer cells to chemotherapeutic drugs by abolishing CAF-mediated drug resistance. SOM230 is an FDA-approved drug (in 2012, for the treatment of Cushing's pituitary tumours) that, despite inducing hyperglycaemia, does not generate any toxicity (Schmid, ; Chan et al, ; Henry et al, ), unlike translation mTOR inhibitors such as RAD001 (Everolimus® Novartis). SOM230 has a high affinity (nanomolar ranges) for all somatostatin receptors, except sst4 (Schmid, ). However, the SOM230 inhibitory effect in CAFs was shown to be specifically mediated through sst1, as demonstrated through sst1 expression knock-down (RNA interference) studies. The mechanisms underlying the high expression of sst1, and elevated PI3K/mTORC1 pathway activation in CAFs, particularly when compared to inactivated α-SMA-negative PaSCs, are currently under investigation. Importantly, CAF treatment with SOM230 does not affect sst1 expression levels, which is a prerequisite for the prospective use of this drug for the long-term treatment of PDAC patients. This result however indicates that SOM230 does not inhibit the translation of all mRNAs in CAFs.

Without this pathway, protein synthesis would ..

21/01/2018 · Lesson on Hedgehog protein synthesis and cellular release

Skeletal muscle protein turnover is regulated by endocrine hormones, nutrients, and inflammation. α-Lipoic acid (ALA) plays an important role in energy homeostasis. Therefore, the aim of this study was to investigate the effects of ALA on protein synthesis in skeletal muscles and reveal the underlying mechanism. ALA (25 μM) significantly increased the protein synthesis and phosphorylation of Akt, mTOR, and S6 in C2C12 myotubes with attenuated phosphorylation of AMPK, Ikkα/β, and eIF2α. Intraperitoneal injection of 50 mg/kg ALA also produced the same results in mouse gastrocnemius. Both the PI3K (LY294002) and mTOR (rapamycin) inhibitors abolished the effects of ALA on protein synthesis in the C2C12 myotubes. However, AICAR (AMPK agonist) failed to block the activation of mTOR and S6 by ALA. ALA increased TLR2 and MyD88 mRNA expression in the C2C12 myotubes. TLR2 knockdown by siRNA almost eliminated the effects of ALA on protein synthesis and the Akt/mTOR pathway in the C2C12 myotubes. Immunoprecipitation data showed that ALA enhanced the p85 subunit of PI3K binding to MyD88. These findings indicate that ALA induces protein synthesis and the PI3K/Akt signaling pathway by TLR2.

Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome-triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E-BP1 regulatory pathway which we found highly activated in primary cultures of α-SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E-BP1 pathway and the resultant synthesis of secreted proteins including IL-6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine-induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1-directed pharmacological compounds represents an anti-stromal-targeted therapy with promising chemosensitization potential.

what is the path of protein synthesis? | Yahoo Answers

30/10/2006 · What is the path of protein synthesis

To verify this possibility, the chemosensitivity of different pancreatic cancer cell lines was tested in the presence of CM from CAFs or PaSCs that had been grown for 48 h without foetal calf serum (passages 3 to 8) (Fig). An MTT viability assay demonstrated that the cytotoxic action of gemcitabine (Fig), 5-fluorouracil (5FU) or oxaliplatin (Supplementary Fig S2A–B) on pancreatic Panc-1 cancer cells was completely reversed upon Panc-1 cell co-treatment with CM from CAFs, whereas CM from PaSCs did not provide any chemoprotection. In addition, heating CM to 95°C significantly diminished the chemoprotective capacity, suggesting a significant role of proteins in the process (Supplementary Fig S2C).

In PDACs, the abundant fibrotic stroma produced by CAFs constitutes a mechanical scaffold and a physical barrier against the effective delivery of therapeutic agents (Olive et al, ). Antifibrotic therapy therefore appears promising for the treatment of PDAC, although it is a ‘symptomatic’ and non-selective strategy (Erkan, ). Besides secreting fibrillar ECM components, CAFs secrete soluble growth, angiogenic and inflammatory factors, that engage in cancer and other stromal cell survival and metastatic and angiogenic signalling that promotes tumour growth and invasion (Hwang et al, ; Vonlaufen et al, ). Importantly, the signals stimulated by CAFs in cancer cells are redundant to those targeted by therapies, conferring innate resistance (Apte et al, ; Erkan, ). Since CAFs are master ‘secretors’ of soluble and insoluble factors which form these specific stromal features, we hypothesized that targeting CAF secretion would represent a specific therapeutic option for PDAC. Further understanding of the mechanisms governing CAF secretion may assist in the development of novel therapies to overcome CAF-triggered drug resistance. In this manuscript, the critical role of mTOR/4E-BP1 signalling pathway activation in promoting protein synthesis and secretion in CAFs has been elucidated. Additionally, a specific pharmacological strategy to stop protein synthesis and secretion through inhibition of this pathway in CAFs is proposed as a novel promising strategy, which has to be used in combination with chemotherapy in the treatment of PDAC.

What path does a protein in the secretory pathway take, starting from its site of synthesis
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SOM230 does not have any direct inhibitory effect on pancreatic cancer cell features in vitro (proliferation, migration or invasion) (data not shown). No expression of sst1 was observed in any of the five pancreatic cancer cell lines (Panc-1, BxPC-3, Capan-1, MIA PaCa-2, CFPAC-1), or in pancreatic cancer cells from forty-two human PDACs, as assessed by Western blot or immunofluorescence analyses, respectively, using an anti-sst1 antibody, the specificity of which had been validated here. In orthotopic co-xenograft cancer cell + CAF and PDX (patient-derived tumour sub-cutaneous xenograft) experiments, SOM230 treatment alone diminished CAF activation but was not able to affect cancer cell proliferation or apoptosis. In PDX experiments, SOM230 significantly decreased tumour progression, probably due to a reduction in the rate of collagen deposition by CAFs (Masson's trichrome staining). Therefore, our results show that the inhibitory effect of SOM230 on CAF secretions is insufficient to affect tumour growth, but is sufficient to provide potent chemosensitization, bypassing pancreatic cancer cell resistance to gemcitabine. Our hypothesis for these differences is that pancreatic cancer cells may be able to adapt in vivo to the decrease in CAF-derived growth signals induced by SOM230 treatment, but not to the absence of CAF-derived chemoprotective factors. Therefore, these results emphasize the role of CAFs as critical chemoprotective cell partners to pancreatic cancer cells.

A tagged protein is then sucked into a large cellular machine ..

Therapeutic strategies aimed at symptomatically targeting the PDAC pro-tumoural fibrotic scaffold using non-selective enzymes that degrade it (Provenzano et al, ; Jacobetz et al, ), CD-40-educated macrophages that alter tumour stroma (Beatty et al, ), or nanoparticle albumin-bound chemotherapy (Von Hoff et al, ), showed promising results in pre-clinical models because they led to better chemotherapy delivery and uptake through stromal depletion and enhanced vascularization (Heinemann et al, ). Several clinical trials in which various forms of antifibrotic therapies are applied concomitantly with gemcitabine are now recruiting patients. Caution has however to be taken regarding these non-selective antifibrotic therapies since breaking down the stromal wall may increase dissemination of cancer cells (Erkan, ). Other options to target CAFs in PDAC consist of treatments which reduce CAF proliferation, including hedgehog pathway inhibitors, retinoic acid or pirfenidone which reduce stroma abundance in PDAC models (Olive et al, ; Froeling et al, ; Kozono et al, ), or vitamin D receptor activation which reprogrammes CAFs to a quiescent phenotype (Sherman et al, ). Although very promising in initial preclinical studies (Olive et al, ), targeting the proliferation of PDAC-associated fibroblasts using hedgehog pathway inhibitors has failed in phase II trials (Amakye et al, ). Shh genetic deletion, or chronic treatment with a hedgehog inhibitor, in mice presenting with an intra-pancreatic mutation of Kras and p53, accelerated pancreatic tumour progression and reduced survival, with tumours presenting a poorly differentiated histology and increased vascularity (Rhim et al, ). Recent findings report that depending on the dosage of hedgehog signalling in the tumour (high, low/absent or only decreased by inhibitors), tumour growth is accelerated, arrested or enhanced, respectively, through increased angiogenesis, probably explaining the unexpected non-efficacy of hedgehog inhibitors in patients (Mathew et al, ).

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