Session 4.3: Backer
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1) The document discusses the relationship between mTORC1, a protein kinase complex that regulates cell growth, and the endosomal system. 2) It reports that disrupting the endosomal system by activating Rab5, which inhibits early to late endosomal conversion, prevents mTORC1 from being activated by amino acids. 3) The findings suggest that proper trafficking through the endosomal system is required for amino acids to induce mTORC1 translocation and activation.
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The document summarizes work using synthetic protein scaffolds to study the effect of enzyme scaffolding on the rate of product formation in biochemical pathways. Key points:
- Proteins involved in the resveratrol pathway were successfully expressed and purified except CFP proteins due to contamination.
- A FRET assay will be used to determine if scaffold assembly was successful by observing higher YFP and lower CFP fluorescence intensities.
- Understanding how kinetic parameters of enzymes in pathways relate to increased product yield from scaffolding is important for biofuels and pharmaceuticals.
- Future work will purify remaining proteins, perform FRET and HPLC to quantify resveratrol production from scaffolded versus non-sc
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1) Removal of amino acids from Chinese hamster ovary cells rapidly decreased the activity of p70 S6 kinase and increased binding of initiation factor eIF4E to its inhibitory regulator 4E-BP1.
2) Resupplying amino acids reversed these effects, and this reversal was blocked by rapamycin and inhibitors of phosphatidylinositol 3-kinase, implying a role for this kinase in linking amino acids to p70 S6 kinase regulation.
3) Amino acid withdrawal did not activate the stress-activated kinases p38 MAP kinase or JNK, and did not affect activity of the kinase PKB, indicating amino acids regulate p70 S6 kinase through a
Abstract
This study explored methods for detecting pyrrole-peptide and pyrrole-protein adducts as potential biomarkers for ragwort poisoning in horses. Initial experiments showed that different reagents can distinctly modify proteins and have varying specificity for cysteine residues. Reaction of pyrrole with the amino acid cysteine and model peptides showed association with cysteine sulphydryl groups. Reaction of pyrrole with equine blood proteins induced cross-linking of abundant plasma proteins like albumin and fibrinogen. An alternative technique used silver nitrate to cleave pyrrole from sulphydryl groups, followed by gas chromatography-mass spectrometry to detect pyrroles in liver and blood from rats dosed with pyrrole and
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This document discusses receptor tyrosine kinases (RTKs) and their role in signal transduction. It notes that RTKs are membrane proteins that transmit signals from outside the cell into the cell. When a ligand binds to an RTK, it causes the receptor to dimerize and phosphorylate itself. This activated RTK then transmits the signal by activating downstream proteins like Ras via GRB2 and Sos. Ras further transduces the signal to serine/threonine kinases and transcription factors, ultimately leading to responses in the cell like growth, differentiation, and metabolism. There are 58 known RTK subfamilies involved in various cellular processes.
Cross talk between signalling pathway
Biological crosstalk refers to instances in which one or more components of one signal transduction pathway affects another.
This can be achieved through a number of ways with the most common form being crosstalk between proteins of signaling cascades.
In these signal transduction pathways, there are often shared components that can interact with either pathway.
A more complex instance of crosstalk can be observed with transmembrane crosstalk between the extracellular matrix (ECM) and the cytoskeleton.
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The document summarizes research findings about the role of Protein Kinase C ζ (PKCζ) in the regulation of translation. The key findings are:
1. PKCζ affects translation independently of the mTOR pathway and through its interaction with the scaffold protein RACK1 on ribosomes.
2. PKCζ associates with ribosomes upon activation and binds specifically to the 40S ribosomal subunit.
3. Disrupting the interaction between PKCζ and RACK1 or knocking down RACK1 impairs translation, suggesting their interaction on ribosomes is required for PKCζ to modulate translation.
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The document summarizes work using synthetic protein scaffolds to study the effect of enzyme scaffolding on the rate of product formation in biochemical pathways. Key points:
- Proteins involved in the resveratrol pathway were successfully expressed and purified except CFP proteins due to contamination.
- A FRET assay will be used to determine if scaffold assembly was successful by observing higher YFP and lower CFP fluorescence intensities.
- Understanding how kinetic parameters of enzymes in pathways relate to increased product yield from scaffolding is important for biofuels and pharmaceuticals.
- Future work will purify remaining proteins, perform FRET and HPLC to quantify resveratrol production from scaffolded versus non-sc
9693128
1) Removal of amino acids from Chinese hamster ovary cells rapidly decreased the activity of p70 S6 kinase and increased binding of initiation factor eIF4E to its inhibitory regulator 4E-BP1.
2) Resupplying amino acids reversed these effects, and this reversal was blocked by rapamycin and inhibitors of phosphatidylinositol 3-kinase, implying a role for this kinase in linking amino acids to p70 S6 kinase regulation.
3) Amino acid withdrawal did not activate the stress-activated kinases p38 MAP kinase or JNK, and did not affect activity of the kinase PKB, indicating amino acids regulate p70 S6 kinase through a
Abstract
This study explored methods for detecting pyrrole-peptide and pyrrole-protein adducts as potential biomarkers for ragwort poisoning in horses. Initial experiments showed that different reagents can distinctly modify proteins and have varying specificity for cysteine residues. Reaction of pyrrole with the amino acid cysteine and model peptides showed association with cysteine sulphydryl groups. Reaction of pyrrole with equine blood proteins induced cross-linking of abundant plasma proteins like albumin and fibrinogen. An alternative technique used silver nitrate to cleave pyrrole from sulphydryl groups, followed by gas chromatography-mass spectrometry to detect pyrroles in liver and blood from rats dosed with pyrrole and
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This document discusses phospholipase C (PLC) and its role in neutrophil degranulation and T cell cytotoxicity. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. PLC signaling is required for lytic granule polarization and effective T cell killing through T cell antigen receptor activation. Neutrophil degranulation involves increases in calcium and PLC production of phosphatidylinositol, which is essential for granule exocytosis. Different intracellular signaling pathways regulate the release of primary, secondary, and tertiary granules from neutroph
Tyrosine kinase linked receptors
This document discusses receptor tyrosine kinases (RTKs) and their role in signal transduction. It notes that RTKs are membrane proteins that transmit signals from outside the cell into the cell. When a ligand binds to an RTK, it causes the receptor to dimerize and phosphorylate itself. This activated RTK then transmits the signal by activating downstream proteins like Ras via GRB2 and Sos. Ras further transduces the signal to serine/threonine kinases and transcription factors, ultimately leading to responses in the cell like growth, differentiation, and metabolism. There are 58 known RTK subfamilies involved in various cellular processes.
Cross talk between signalling pathway
Biological crosstalk refers to instances in which one or more components of one signal transduction pathway affects another.
This can be achieved through a number of ways with the most common form being crosstalk between proteins of signaling cascades.
In these signal transduction pathways, there are often shared components that can interact with either pathway.
A more complex instance of crosstalk can be observed with transmembrane crosstalk between the extracellular matrix (ECM) and the cytoskeleton.
FISV2008
The document summarizes research findings about the role of Protein Kinase C ζ (PKCζ) in the regulation of translation. The key findings are:
1. PKCζ affects translation independently of the mTOR pathway and through its interaction with the scaffold protein RACK1 on ribosomes.
2. PKCζ associates with ribosomes upon activation and binds specifically to the 40S ribosomal subunit.
3. Disrupting the interaction between PKCζ and RACK1 or knocking down RACK1 impairs translation, suggesting their interaction on ribosomes is required for PKCζ to modulate translation.
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This document discusses tyrosine kinase inhibitors (TKIs), a class of targeted cancer drugs. It begins by introducing protein kinases and their role in cell signaling. There are two main categories of protein kinases - those that phosphorylate tyrosine residues and those that phosphorylate serine and threonine residues. Tyrosine kinases function as on/off switches in many cellular functions by adding phosphate groups to tyrosine residues on proteins. The document then discusses the different types of tyrosine kinases and how they can become mutated and cause unregulated cell growth leading to cancer. It describes targeted therapy and TKIs as targeted drugs that block specific molecules needed for tumor growth. The final sections provide examples of approved TKIs
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In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus. The entire process is called gene expression.
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The document discusses research on using phospholipase A2 (PLA2) antibodies to protect cells from radiation damage. PLA2 is an enzyme that releases fatty acids from phospholipids in cell membranes. Exposure to ionizing radiation activates PLA2, leading to cell death. The research proposal is to test whether antibodies that block PLA2 can inhibit this activation and thereby reduce the toxic effects of radiation exposure and increase radiation survival. Blocking PLA2 with antibodies may provide a new radiation protection strategy and tool for diagnosing and monitoring acute radiation sickness.
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1. The study investigates the role of the regulatory subunit SUR-6 in the protein phosphatase 2A (PP2A) complex and its effect on muscle protein degradation in the nematode C. elegans.
2. Experiments show that RNAi knockdown of SUR-6 prevents severe muscle degradation in a daf-2 mutant strain at 25°C, suggesting SUR-6 is required for PP2A to activate the MAPK pathway and promote degradation.
3. The findings indicate SUR-6 allows PP2A to target and activate RAF kinase by removing inhibitory phosphates placed by AKT, thus positively regulating muscle protein degradation.
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Non-receptor tyrosine kinases (nRTKs) are cytoplasmic enzymes that catalyze the transfer of phosphate groups from ATP to tyrosine residues in proteins. Unlike receptor tyrosine kinases, nRTKs lack extracellular and transmembrane domains. nRTKs contain two domains - a catalytic domain consisting of two lobes that facilitate ATP transfer, and a protein-protein interaction domain containing SH2 and SH3 subdomains. There are several families of nRTKs including Src, Abl, Syk, Jak, and Zap70 that perform critical functions in immune cell signaling through T cell and B cell receptors. Inhibitors of overactive nRTKs show promise for cancer treatment by blocking intracellular tumor processes.
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The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
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Introduction of different types of primary and secondary messengers including hydrophilic, hydrophobic and gaseous.
it also describes the intracellular pathway of cyclic amp, jak stat and mapk in a very simple language.
Second messengers cAMP and cGMP
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This document provides an overview of receptor tyrosine kinases (RTKs) and their signaling pathways. It begins with an introduction to cell signaling and the different types of receptors, focusing on protein tyrosine kinases. RTKs are described as high-affinity cell surface receptors that activate upon ligand binding and recruit signal transduction proteins. The mechanisms of RTK activation and recruitment of proteins like Ras are explained. It further details how RTK activation of Ras leads to the Ras/MAP kinase signaling cascade, which regulates processes like cell division and differentiation. The relationships between different signaling pathways that can activate the Ras/MAP kinase cascade are also noted.
Transcripion
1. There are four main classes of RNA: ribosomal RNA, messenger RNA, transfer RNA, and small nuclear RNA.
2. Transcription involves three stages - initiation, elongation, and termination. It uses DNA as a template to produce RNA.
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This document discusses co-translational and post-translational modifications of proteins. It begins with an introduction to protein modification and defines co-translational and post-translational modifications. It then covers various co-translational modifications including regulation of translation, protein folding, and enzymes that catalyze protein folding. Post-translational modifications discussed include protein cleavage, glycosylation, addition of GPI anchors, ubiquitination, and phosphorylation. The document provides examples and details for many of the modification processes.
Prokaryotic translation machinery by kk
Introduction
Definition
Factors required for Translation
Formation of aminoacyl t-RNA
1)Activation of amino acid
2) Transfer of amino acid to t-RNA
Translation involves following steps:-
1)Initiation
2)Elongation
3)Termination
Conclusion
Reference
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MEHP targets signaling in spermatogonial stem cells. The study found that MEHP affects ERK signaling and gene expression in a spermatogonial stem cell line. Specifically, MEHP affected Fos expression and proliferation but did not alter differentiation-related gene expression. MEHP also altered expression of stem cell markers PLZF and GFRA1 as well as differentiation marker KIT. MEHP decreased GDNF production by Sertoli cells, which could impact stem cell maintenance. Transplantation experiments will determine if MEHP exposure alters stem cell function in vivo.
Venters Molecular and Cellular Biology 2011 2253-2261
This study investigates the contributions of conserved regions of the essential yeast protein Mot1 to genome-wide gene regulation and transcription factor (TBP) recruitment. The researchers used a transient replacement strategy to delete conserved regions of Mot1 and analyzed the effects on gene expression and TBP occupancy. They found that the four conserved regions of Mot1 are generally required for all Mot1-regulated genes. Deletion of the ATPase region specifically altered transcriptional dependence for some genes and caused TBP to redistribute away from Mot1-inhibited genes and to other normally Mot1-independent genes. This suggests that Mot1 uses ATP hydrolysis to redistribute accessible TBP between sites of high and low intrinsic preference.
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The most recurrent cause of cancer-related deaths worldwide in women is the breast cancer. The key to diagnosis is early prediction and a curable stage but still treatment remains a great clinical challenge. Origin of the Problem: A number of studies have been carried out for the treatment of breast cancer which includes the targeted therapies and increased survival rates in women. Essential PI3K/mTOR signaling pathway activation has been observed in most breast cancers. The cell growth and tumor development in such cases involves phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) complex intracellular pathway. Hypothesis: Through preclinical and clinical trials, it has been observed that there are a number of other inhibitors of PI3K/Akt/mTOR pathway, which either alone or in combination with cytotoxic agents can be used for endocrine therapies. Conclusions: Structure and regulation/deregulation of mTOR provides a greater insight into the action mechanism. Also through this review, one could easily scan first and second generation inhibitors for PI3K/Akt/mTOR pathway besides targeted therapies for breast cancer and the precise role of mTOR.
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Translation and its regulation post translational modification
In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus. The entire process is called gene expression.
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The document discusses research on using phospholipase A2 (PLA2) antibodies to protect cells from radiation damage. PLA2 is an enzyme that releases fatty acids from phospholipids in cell membranes. Exposure to ionizing radiation activates PLA2, leading to cell death. The research proposal is to test whether antibodies that block PLA2 can inhibit this activation and thereby reduce the toxic effects of radiation exposure and increase radiation survival. Blocking PLA2 with antibodies may provide a new radiation protection strategy and tool for diagnosing and monitoring acute radiation sickness.
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1. The study investigates the role of the regulatory subunit SUR-6 in the protein phosphatase 2A (PP2A) complex and its effect on muscle protein degradation in the nematode C. elegans.
2. Experiments show that RNAi knockdown of SUR-6 prevents severe muscle degradation in a daf-2 mutant strain at 25°C, suggesting SUR-6 is required for PP2A to activate the MAPK pathway and promote degradation.
3. The findings indicate SUR-6 allows PP2A to target and activate RAF kinase by removing inhibitory phosphates placed by AKT, thus positively regulating muscle protein degradation.
Non receptor tyrosine kinases
Non-receptor tyrosine kinases (nRTKs) are cytoplasmic enzymes that catalyze the transfer of phosphate groups from ATP to tyrosine residues in proteins. Unlike receptor tyrosine kinases, nRTKs lack extracellular and transmembrane domains. nRTKs contain two domains - a catalytic domain consisting of two lobes that facilitate ATP transfer, and a protein-protein interaction domain containing SH2 and SH3 subdomains. There are several families of nRTKs including Src, Abl, Syk, Jak, and Zap70 that perform critical functions in immune cell signaling through T cell and B cell receptors. Inhibitors of overactive nRTKs show promise for cancer treatment by blocking intracellular tumor processes.
Seminario molecular[1] listo
This study explores how aristolochic acid (AA) affects leptin production in kidney fibroblasts. The researchers found that AA treatment increased leptin secretion and expression in kidney fibroblasts in a time and dose-dependent manner. AA was found to activate C/EBPα and the PI3K/Akt pathway to upregulate leptin expression. Knocking down C/EBPα or inhibiting the PI3K/Akt pathway blocked AA-induced leptin expression, demonstrating these factors are involved in AA's effects on leptin. Overall, the results suggest AA promotes renal fibrosis by stimulating leptin production in fibroblasts through C/EBPα and PI3K/Akt signaling
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The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
secondary messengers and intracellular signaling
Introduction of different types of primary and secondary messengers including hydrophilic, hydrophobic and gaseous.
it also describes the intracellular pathway of cyclic amp, jak stat and mapk in a very simple language.
Second messengers cAMP and cGMP
Second messengers are intracellular signaling molecules that are released within cells in response to extracellular first messengers like hormones and neurotransmitters. They amplify and propagate intracellular signals. Examples include cyclic AMP (cAMP), cyclic GMP (cGMP), inositol trisphosphate, and calcium. cAMP and cGMP are produced from ATP and GTP by adenylate and guanylate cyclases, respectively. They activate downstream effector proteins like protein kinase A and G. This leads to phosphorylation of various target proteins and physiological responses like metabolism, gene expression, cell survival, proliferation and apoptosis. The document discusses the mechanisms, targets, functions and therapeutic applications of cAMP and cGMP second messenger systems in detail.
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CD47 plays a role in nitric oxide signaling and angiogenesis by interacting with other proteins like thrombospondin-1 and vascular endothelial growth factor receptor-2. The document discusses experiments attempting to characterize CD47's role by tagging it with BirA to identify interacting proteins, expressing a truncated version of thrombospondin-1 to activate CD47 and measure calcium responses, and cloning CD47-BirA into a vector for expression. Preliminary results were inconclusive, suggesting the calcium assay and thrombospondin-1 purification need further optimization to understand CD47's signaling mechanisms.
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This document summarizes a student's research project analyzing genetic variants found in patients diagnosed with Acrodysostosis. 17 variants were identified in the PRKAR1A and PDE4D genes, including 2 rare missense variants, L312F and L319P, that were predicted to be pathogenic. The student hypothesized that these variants could disrupt cAMP signaling and cause Acrodysostosis by altering the structure and function of the PDE4D protein involved in degrading cAMP. The identification of pathogenic variants provided evidence that some Acrodysostosis diagnoses may have been misclassified.
Deeksha
This document provides an overview of receptor tyrosine kinases (RTKs) and their signaling pathways. It begins with an introduction to cell signaling and the different types of receptors, focusing on protein tyrosine kinases. RTKs are described as high-affinity cell surface receptors that activate upon ligand binding and recruit signal transduction proteins. The mechanisms of RTK activation and recruitment of proteins like Ras are explained. It further details how RTK activation of Ras leads to the Ras/MAP kinase signaling cascade, which regulates processes like cell division and differentiation. The relationships between different signaling pathways that can activate the Ras/MAP kinase cascade are also noted.
Transcripion
1. There are four main classes of RNA: ribosomal RNA, messenger RNA, transfer RNA, and small nuclear RNA.
2. Transcription involves three stages - initiation, elongation, and termination. It uses DNA as a template to produce RNA.
3. Eukaryotic transcription requires RNA polymerase and other transcription factors to initiate transcription from a promoter region on DNA. The transcription process includes initiation, elongation, and termination.
Unit 9 Dna Translation And Mutation
Translation converts mRNA messages into polypeptides through the use of codons, anticodons, transfer RNA (tRNA), and ribosomes. A codon is a sequence of three nucleotides that codes for a specific amino acid. The ribosome helps form a polypeptide bond between amino acids specified by mRNA codons. Gene expression is regulated at transcription and RNA processing in both prokaryotes and eukaryotes. Mutations are changes in DNA that may or may not affect phenotype and can occur through replication errors, mutagens like UV rays, or during chromosomal crossover.
Co and post translationational modification of proteins
This document discusses co-translational and post-translational modifications of proteins. It begins with an introduction to protein modification and defines co-translational and post-translational modifications. It then covers various co-translational modifications including regulation of translation, protein folding, and enzymes that catalyze protein folding. Post-translational modifications discussed include protein cleavage, glycosylation, addition of GPI anchors, ubiquitination, and phosphorylation. The document provides examples and details for many of the modification processes.
Prokaryotic translation machinery by kk
Introduction
Definition
Factors required for Translation
Formation of aminoacyl t-RNA
1)Activation of amino acid
2) Transfer of amino acid to t-RNA
Translation involves following steps:-
1)Initiation
2)Elongation
3)Termination
Conclusion
Reference
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Translation and its regulation post translational modification
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In prokaryotes:
- Genes are organized into operons that are regulated by repressor proteins binding to operator sequences. This allows for coordinated expression of genes in response to environmental conditions, as seen in the lac and tryptophan operons.
In eukaryotes:
- Gene expression is influenced by chromatin structure and modifications to histone proteins like acetylation and methylation that make DNA more or less accessible. DNA methylation can also silence genes. Post-transcriptional mechanisms further regulate gene expression.
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Carnosic acid (CA) is a bioactive molecule found in rosemary and sage plants that has antioxidant and anticancer properties. This study examined the effects of CA on glioblastoma (GBM) cells. The results showed that CA decreased cell survival of GBM cells in a dose-dependent manner by inducing G2 cell cycle arrest and apoptosis. CA modulated key signaling pathways involved in proliferation and survival, including decreasing phosphorylation of STAT3 and AKT. It also promoted proteasomal degradation of proteins important for proliferation like SOX2 and GFAP. However, CA failed to degrade MYC. In conclusion, CA has potential as an anticancer agent for GBM but its clinical potential is limited as it cannot
Teloomerase Research Paper
Here are the key biological and physiological mechanisms involved in genetically modified crops:
1. Gene insertion: The desired gene is isolated and inserted into the plant's genome using recombinant DNA technology. Common methods include Agrobacterium-mediated transformation or direct DNA delivery via particle bombardment.
2. Promoter selection: The inserted gene is regulated by selecting an appropriate promoter that controls the timing and location of gene expression. Constitutive promoters allow continuous expression in all tissues, while tissue-specific promoters restrict expression.
3. Protein expression: The inserted gene is transcribed and translated inside the plant cell, resulting in the production of a novel protein or trait. For example, Bt crops express insecticidal Cry proteins from Bacillus thuring
Dr. Goy MCL
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The document summarizes a presentation about using BioMAP human cell systems to analyze the mechanisms of drug compounds, including PPAR agonists. It describes how BioMAP uses primary human cells in disease-like conditions to generate quantitative readouts that can discriminate between clinical compounds. Profiling of PPAR agonists using BioMAP revealed both shared and unique anti-inflammatory, tissue remodeling, and prostaglandin pathway effects. Differential activities suggested priorities for therapeutic areas and potential side effects.
Na f activates map ks and induces apoptosis in odontoblast-like
The study examined the effects of sodium fluoride (NaF) on odontoblast-like MDPC-23 cells. The researchers found that NaF exposure induced apoptosis in a dose-dependent manner through several markers. NaF activated the mitogen-activated protein kinases (MAPKs) JNK and p38, and induced two peaks in ERK phosphorylation. Inhibition of JNK suppressed NaF-induced apoptosis, while inhibition of p38 and ERK had lesser effects, suggesting NaF-induced apoptosis depends primarily on JNK signaling.
Pathways of intracellular signal transduction
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Lac Operon
The document summarizes gene regulation in prokaryotes and eukaryotes. In prokaryotes, genes are organized into operons that are regulated by repressor or inducible systems like the tryptophan and lactose operons. The tryptophan operon is turned off by a repressor when tryptophan levels are high. The lactose operon is induced when lactose is present and glucose levels are low. In eukaryotes, gene expression is controlled through chromatin structure, pre-transcriptional and post-transcriptional regulation involving transcription factors and RNA processing, and pre-translational and post-translational regulation involving translation initiation, protein processing and degradation.
Thesis defense
PUF proteins are able to repress translation in vitro. Puf5p from yeast can repress expression of a reporter mRNA in a cell-free translation assay. Puf5p regulates many mRNAs, but binding alone is not sufficient for regulation. Puf5p uses two mechanisms to repress translation of the HO mRNA - one dependent on spacing from the ORF to the 3'UTR, and one dependent on the poly(A) binding protein Pab1p. Pab1p is required for Puf5p repression when oligo(A) tracts are present in the 3'UTR.
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Session 4.3: Backer
- 1. mTORC1 and the Endosomal System JON BACKER DEPT. OF MOLECULAR PHARMACOLOGY ALBERT EINSTEIN COLLEGE OF MEDICINE BRONX, NEW YORK
- 2. Cell Growth The mTOR Protein Kinase Ma and Blenis, Nat. Rev. Mol. Biol. 2009 Nutrients
- 3. LKB1 PTEN TSC1 TSC2
- 4. Guertin and Sabatini, Cancer Cell 2007 mTOR and Angiogenesis
- 5. Dancey et al., Sem. Oncology 2009
- 6. Dancey et al., Sem. Oncology 2009
- 7. Dancey et al., Sem. Oncology 2009
- 8. Liu et al, Nat. Rev. Drug Discovery 2009 Anti-Signaling Drugs in Clinical Trials
- 9. S6K1 TORC1 Rheb(GTP) TSC1/2 Akt Insulin 4EBP1 mTOR and Translational Control Ribosomal S6 Translation initiation Amino Acids hVps15/ hVps34 Glucose AMPK
- 10. Vps34 Vps15 Rab5 GTP PI[3]P Christoforidis et al., Nature Cell Biol. 1999 Murray et al., Traffic (2002) hVps34 Regulates Early Endosomal Fusion P P hVps34 Class III PI[3]P PX FYVE hVps15
- 11. Former Labmates Maya Byfield James Murray Gary Gerfen Mark Girvin Ana Maria Cuervo Invasion and Motility PO1 Anne Bresnick Diane Cox John Condeelis Jeff Pollard Jeff Segall Richard Stanley Roger Williams (Cambridge) Somasekar Seshagiri (Genentech) Tom Neufeld (Univ. Minnesota) Kevan Shokat (USCF) Bart Vanhaesebroeck (London Sch. Med.) Funding: NIH, NCI, ADA
- 12. CCV EE RC LE Lysosome TGN Plasma Membrane 2 1 3 4 5 5 6 Is mTORC1 Inhibited by Disruption of the Endocytic System? Mutant Effect 1) Rab5 CA Inhibit 2) Eps15 No Effect 3) Rab11 Mutants No Effect 4) Rab7 Mutants No Effect 5) PIKFyve DN No Effect 6) SKD1 DN No Effect
- 13. Constitutive activation of Rab5 blocks mTORC1/S6K1 activation AA Stimulated S6K1 in HEK293T Flinn et al. Mol. Biol. Cell. (2010)
- 14. CCV EE RC LE Lysosome TGN Plasma Membrane 2 1 3 4 5 5 6 Is mTORC1 Inhibited by Disruption of the Endocytic System? Mutant Effect 1) Rab5 CA Inhibit 2) Eps15 No Effect 3) Rab11 Mutants No Effect 4) Rab7 Mutants No Effect 5) PIKFyve DN No Effect 6) SKD1 DN No Effect
- 15. Rab5CA blocks early/late endosomal conversion producing hybrid early/late endosomes Flinn et al. Mol. Biol. Cell. (2010)
- 16. Blocking early/Late endosomal conversion by hVps39 KnDn inhibits mTORC1/S6K1 EEA1 LAMP2 5 micron 5 micron 20 micron 20 micron Hela Flinn et al. Mol. Biol. Cell. (2010) S6K1 Phosphorylation
- 17. Sancak et al, Science. 2008 mTOR Activation by Amino Acids Requires Rag-mediated Endosomal Translocation
- 18. Amino Acids Induce mTOR translocation Flinn et al. Mol. Biol. Cell. (2010)
- 19. Rab5CA leads to mTOR localization to hybrid early/late endosomes Flinn et al. Mol. Biol. Cell. (2010)
- 20. mTOR, hVps34 and the Endosomal System EE LE MVB Lys Rab5 V34 V15 Rab7 V34 V15 HOPS Lamp2 Rheb Rab5
- 21. mTOR, hVps34 and the Endosomal System EE LE MVB Lys Rab5 V34 V15 Rab7 V34 V15 Rheb mTOR (off) mTOR (on) Amino Acid Starved Amino Acid Replete Lamp2 HOPS Rab5
- 22. MVB Lys mTOR (off) Amino Acid Replete EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System LAMP2
- 23. MVB Lys Rheb mTOR (off) EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System X LAMP2
- 24. Activation of Endogenous Rheb Does not Rescue Rab5CA Inhibition of mTOR TSC2-/- MEFs Flinn et al. Mol. Biol. Cell. (2010)
- 25. MVB Lys mTOR (off) EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System X TSC1/2 X Rheb LAMP2
- 26. MVB Lys Rheb mTOR (off) EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System Rheb Rheb Rheb Rheb Rheb Rheb Rheb Rheb Rheb X LAMP2
- 27. Endosomal Alkalinization blocks mTOR Activation
- 28. HEK293T AA Stimulated S6K1 Exogenous Rheb renders mTORC1 insensitive to ConA/NH4Cl AA:
- 29. Increasing intraluminal endosomal pH inhibits mTORC1 signaling
- 30. Endosomal Alkalinization inhibits amino acid-stimulated mTORC1/S6K1 signaling Hela AA Stimulated S6K1