The document discusses prion diseases and the protein-only hypothesis of pathogenesis. It summarizes the structures of cellular and scrapie prion protein and describes techniques used to study prion protein aggregates, including EPR spectroscopy. The document outlines research showing recombinant prion protein forms amyloid fibrils with a parallel in-register beta-sheet structure between residues 160-220. Both denaturing and native conditions produce similar amyloid fibrils, but buffer conditions can lead to structurally distinct fibrils. Substitution of single residues is also shown to produce different amyloid structures.
USMLE Step 1 Molecular Biology and Biochemistry reviewAbril Santos
This is a review for the USMLE Step 1 exam for Molecular Biology and Biochemistry.
It contains anything you need to know for your exam in pictures, tables and diagrams.
Presentation made by Dr. Paul Taylor on October 30, 2015 at the Alzforum-hosted live webinar titled "Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?"
More information and the recording of the session available at http://www.alzforum.org/webinars/fluid-business-could-liquid-protein-herald-neurodegeneration
The document discusses the evolution of chromatin proteins and prediction of novel factors in chromatin dynamics. It summarizes that chromatin proteins show increasing complexity with organismal complexity. Analysis of protein domain architectures can reveal lineage-specific connections and predict novel functions. For example, it predicts that a family of proteins called DBC1 may integrate regulatory inputs from NAD metabolites. The document also predicts that some two-headed enzymes may exist that can modify both lysines and glutamates on histone proteins.
Chapter 5 -repair or radiation damage and dose-rate effect - jtlJohn Lucas
The document summarizes various pathways for repairing DNA damage from radiation: base excision repair removes inappropriate bases; nucleotide excision repair removes bulky adducts like pyrimidine dimers. Mismatch repair fixes base-base mismatches. Non-homologous end joining and homologous recombination repair double-strand breaks, with the former being error-prone and active in G1, and the latter being error-free using a sister chromatid template and most active in G2 phase. Certain syndromes like ataxia-telangiectasia and LIG4 syndrome result from defects in these pathways and cause radiation sensitivity.
The document provides an overview of DNA repair, including:
1) DNA is the only biological macromolecule that is repaired, as spontaneous and environmentally-induced damage occurs daily.
2) There are multiple pathways of DNA repair, including direct reversal, base excision repair, nucleotide excision repair, and double-strand break repair.
3) Defects in DNA repair can lead to genetic disorders and cancer, highlighting the importance of effective repair.
This document discusses the process of translation and protein synthesis. It covers topics like the genetic codon, tRNA, amino acid activation, and the roles of the ribosome. It also discusses inhibitors of protein and RNA synthesis that can act on prokaryotes, both prokaryotes and eukaryotes, or just eukaryotes. Finally, it briefly touches on protein folding, molecular chaperones, protein quality control, and triggering sister chromatid separation. The document is authored by Qurat-ul-Ain, who has a Ph.D. in molecular biology and genetics.
The document summarizes the contemporary method of chemically synthesizing DNA, known as the phosphoramidite method. It describes how this method works by adding nucleotides one by one in an automated DNA synthesizer using a 3' to 5' direction, opposite of natural DNA synthesis. The phosphoramidite method is widely used due to its high yield, purity, and adaptability to automated machines. Single-stranded DNA fragments are synthesized separately and then joined and ligated together to form the full length synthetic gene of interest.
Introduction
What RNA Splicing???
Discovery
Types
Alternative Splicing
Mechanism
Regulatory element And protein
Splicing repression
Splicing activation
Significance
Diseases
Conclusion
Refrences
USMLE Step 1 Molecular Biology and Biochemistry reviewAbril Santos
This is a review for the USMLE Step 1 exam for Molecular Biology and Biochemistry.
It contains anything you need to know for your exam in pictures, tables and diagrams.
Presentation made by Dr. Paul Taylor on October 30, 2015 at the Alzforum-hosted live webinar titled "Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?"
More information and the recording of the session available at http://www.alzforum.org/webinars/fluid-business-could-liquid-protein-herald-neurodegeneration
The document discusses the evolution of chromatin proteins and prediction of novel factors in chromatin dynamics. It summarizes that chromatin proteins show increasing complexity with organismal complexity. Analysis of protein domain architectures can reveal lineage-specific connections and predict novel functions. For example, it predicts that a family of proteins called DBC1 may integrate regulatory inputs from NAD metabolites. The document also predicts that some two-headed enzymes may exist that can modify both lysines and glutamates on histone proteins.
Chapter 5 -repair or radiation damage and dose-rate effect - jtlJohn Lucas
The document summarizes various pathways for repairing DNA damage from radiation: base excision repair removes inappropriate bases; nucleotide excision repair removes bulky adducts like pyrimidine dimers. Mismatch repair fixes base-base mismatches. Non-homologous end joining and homologous recombination repair double-strand breaks, with the former being error-prone and active in G1, and the latter being error-free using a sister chromatid template and most active in G2 phase. Certain syndromes like ataxia-telangiectasia and LIG4 syndrome result from defects in these pathways and cause radiation sensitivity.
The document provides an overview of DNA repair, including:
1) DNA is the only biological macromolecule that is repaired, as spontaneous and environmentally-induced damage occurs daily.
2) There are multiple pathways of DNA repair, including direct reversal, base excision repair, nucleotide excision repair, and double-strand break repair.
3) Defects in DNA repair can lead to genetic disorders and cancer, highlighting the importance of effective repair.
This document discusses the process of translation and protein synthesis. It covers topics like the genetic codon, tRNA, amino acid activation, and the roles of the ribosome. It also discusses inhibitors of protein and RNA synthesis that can act on prokaryotes, both prokaryotes and eukaryotes, or just eukaryotes. Finally, it briefly touches on protein folding, molecular chaperones, protein quality control, and triggering sister chromatid separation. The document is authored by Qurat-ul-Ain, who has a Ph.D. in molecular biology and genetics.
The document summarizes the contemporary method of chemically synthesizing DNA, known as the phosphoramidite method. It describes how this method works by adding nucleotides one by one in an automated DNA synthesizer using a 3' to 5' direction, opposite of natural DNA synthesis. The phosphoramidite method is widely used due to its high yield, purity, and adaptability to automated machines. Single-stranded DNA fragments are synthesized separately and then joined and ligated together to form the full length synthetic gene of interest.
Introduction
What RNA Splicing???
Discovery
Types
Alternative Splicing
Mechanism
Regulatory element And protein
Splicing repression
Splicing activation
Significance
Diseases
Conclusion
Refrences
This document contains summaries of 4 poster presentations from a conference on Alzheimer's disease and dementia research.
The first poster discusses findings that the ER stress transcription factor XBP1s protects against amyloid-beta neurotoxicity in a fruit fly model of Alzheimer's and in human neuroblastoma cells. XBP1s prevents the accumulation of free calcium in the cytosol, explaining its neuroprotective effects.
The second poster reports that amyloid-beta oligomers impair neuronal function and morphology in rat and mouse neuronal cultures and hippocampal slices through a mechanism linked to NMDA receptor signaling and activation of the protein Jacob.
The third poster examines the role of O-GlcNAc glycosylation in hippocampal synaptic
Presentation made by Dr. Simon Alberti on October 30, 2015 at the Alzforum-hosted live webinar titled "Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?"
More information and the recording of the session available at http://www.alzforum.org/webinars/fluid-business-could-liquid-protein-herald-neurodegeneration
The document discusses DNA replication in prokaryotes and eukaryotes. In prokaryotes, replication begins with initiation at the origin of replication (oriC) mediated by DnaA and other proteins. Bidirectional replication forks are established with the help of helicases and DNA polymerases. Elongation involves continuous synthesis of leading and discontinuous synthesis of lagging strands in Okazaki fragments. Termination occurs when the replication forks from opposite directions converge. In eukaryotes, replication is regulated by cyclin-dependent kinases and occurs at multiple origins of replication with similar initiation and elongation processes as prokaryotes.
The document summarizes 4 projects being conducted as part of a PhD program:
1) Studying the effect of G protein lipids on membrane structure using molecular dynamics simulations. Results show the lipids interact differently with the membrane.
2) Examining 2-hydroxy arachidonic acid as a potential anti-inflammatory drug through binding energy and docking analyses with COX enzymes. It may have advantages over arachidonic acid.
3) Investigating how the drug BGP-15 remodels plasma membrane rafts using density profiles. It enhances docking with cholesterol-rich regions and increases order.
4) Developing GRIMD, a system for distributed molecular dynamics simulations across multiple machines
The document discusses several key concepts in molecular biology including DNA, RNA, transcription, translation, and protein synthesis. It explains that DNA is transcribed into RNA which is then translated into proteins. It describes the central dogma of molecular biology and provides more details on processes like transcription, splicing, and translation. It also discusses topics like alternative splicing, RNA editing, and RNA interference.
This document summarizes Federica Campana's doctoral thesis on investigating drug-cell membrane interactions using molecular dynamics simulations. The thesis examines how membrane composition influences the effects of membrane fluidizers and heat shock protein co-inducers. It also analyzes the binding of anti-inflammatory molecules like hydroxyarachidonic acid to cyclooxygenase enzymes. The overall goal is to better understand how drug molecules interact with and modulate lipid bilayer properties at a molecular level.
Genetics is the study of heredity and genetic variation. Key terms include:
- Genotype is the genetic makeup of an organism, phenotype is observable traits.
- Genes hold information to build cells and pass traits to offspring. The human genome contains 25,000-35,000 genes located on 23 chromosome pairs in the nucleus.
- DNA is transcribed to RNA and translated to proteins, which determine an organism's traits. Variations in genes and chromosomes can result in genetic disorders. Common methods to study genetics include karyotyping, analyzing pedigrees, and identifying alleles and mutations. Understanding genetics provides insight into inheritance patterns and human health.
The genetic code is the system by which nucleotide sequences in mRNA determine the amino acid sequences in proteins. The genetic code uses triplets of nucleotides called codons to specify which amino acid will be incorporated into the growing polypeptide chain. There are 64 possible codons but only 20 standard amino acids, so most amino acids have multiple codons. Three codons act as stop signals to end protein synthesis. The genetic code is nearly universal across all life due to its high degree of specificity and redundancy.
The Drosophila protein Rolling Blackout (RBO) is required for synaptic vesicle endocytosis, as mutants show defects in vesicle recycling and endosome formation; RBO localizes to synapses and interacts genetically with the SNARE protein Syntaxin-1A, suggesting it functions in vesicle fusion or membrane uptake during endocytosis; Loss of RBO specifically impacts endocytosis, as evidenced by accumulation of docked vesicles and defects in dye uptake during stimulation.
This document discusses the learning objectives for a lesson on molecular genetics. It covers DNA structure and function, genes, genetic transfer between organisms, and the effects of genetic engineering on society. Specifically, it aims to explain how the human insulin gene can be inserted into bacterial DNA to produce human insulin through fermentation. It also aims to discuss the social and ethical implications of genetic engineering using a real-world example.
This document describes a study that used CRISPR/Cas9 genome editing to generate zebrafish models of human cardiovascular disorders. The researchers introduced specific point mutations into zebrafish genes linked to dilated cardiomyopathy and Cantu syndrome in humans. They characterized the resulting zebrafish lines and found they exhibited cardiac anomalies and cerebral vasodilation similar to the human conditions. High-speed imaging was used to analyze cardiac function and blood flow in the mutant fish. The study demonstrates that CRISPR/Cas9 can be used to precisely model human genetic diseases in zebrafish at the single nucleotide level.
This document summarizes a study that developed a simplified method for purifying the Thermus aquaticus (Taq) DNA polymerase expressed in Escherichia coli. Key steps included:
1) Overproducing the Taq DNA polymerase in E. coli by cloning the Taq gene into an expression vector and inducing high-level expression.
2) Lysing the E. coli cells and heat-treating the lysate to inactivate endogenous nucleases.
3) Precipitating the Taq polymerase from the lysate using polyethyleneimine and eluting it from an ion exchange resin.
4) Obtaining a purified Taq polymerase preparation in a single-day procedure yielding 40-60 mg of
Structure-Function Analysis of POR MutantsAYang999
This document summarizes protein structure and function, with a focus on mutations in the protein cytochrome P450 oxidoreductase (POR). It describes how mutations can alter a protein's amino acid sequence and structure. Specifically, it investigates the S102P and R550Q mutations in POR, which were found in humans. Experiments expressed and purified these mutant POR proteins, and will test their electron transfer activity compared to the wild type protein using a cytochrome c reduction assay. This will help determine if these mutations impair POR function.
The document describes an experiment where disulfide bonds were engineered into a1-antitrypsin (a1-AT) to lock the movement of helix F (hF) and the connecting loop (thFs3A) during protease inhibition. Disulfide bonds were introduced between thFs3A and strands 3A or 5A of the beta-sheet. The disulfide between thFs3A and strand 5A, but not 3A, eliminated inhibitory activity, suggesting displacement from strand 5A is required for inhibition. The disulfide between thFs3A and strand 3A slowed polymerization without affecting inhibition. This provides direct evidence that hF/thFs3A displacement from strand 5
This presentation aims at giving a vivid knowledge about Nucleoli, a sub organelle of Nucleus, its role in protein formation. control, localization ad how specifically it is involved in Single Nucleotide polymorphism. The slide also discusses about the involvement of SNPs in Alzheimer’s Disease.
Regulation of pten activity by its carboxyl terminal autoinhibitoryChau Chan Lao
Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain.
Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan
From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007
目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白,其由403個氨基酸組成,主要分PTPase及C2 domain,C2 domain使PTEN可與細胞膜作用連結。
PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385),這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株,發現這些變異株具有更強的腫瘤抑制能力,但穩定性將降低,這可能是因這些變異株具有更開放結構所致。
本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株,發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro,而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低,因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法,發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用,暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide,發現其可抑制PTEN之催化活性in vitro,而在細胞表現此peptide則會抑制PTEN之membrane localization,磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。
The genetic code contains rules for translating genetic information in DNA into proteins. It uses triplet codons where each codon specifies a single amino acid. The code is degenerate, with more than one codon coding for an amino acid. It is non-overlapping and non-ambiguous, with each codon corresponding to only one amino acid. Translation proceeds continuously in a 5' to 3' direction, with AUG always initiating protein synthesis and specifying methionine.
This document reports on a study that investigated the effects of oxidative damage to mRNA on translation. The researchers found that a single oxidative lesion, 8-oxoguanine (8-oxoG), drastically reduced the rate of peptide bond formation during translation by more than three orders of magnitude, regardless of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of tRNA selection. This suggests that the modification causes the translational machinery to stall. Consistent with these findings, mRNAs containing 8-oxoG were observed to accumulate and associate with polyribosomes in yeast strains where mRNA surveillance mechanisms were compromised, providing evidence that cells have evolved to cope with damaged mRNA.
The document describes several classes of molecular markers used in genetic analysis, including isozymes, RFLPs, RAPDs, AFLPs, microsatellites, and SNPs. Isozymes analyze differences in protein mobility on a gel, while RFLPs, RAPDs, AFLPs detect DNA fragment length polymorphisms. Microsatellites analyze differences in repeat number, and SNPs detect single nucleotide differences. Each method has advantages and disadvantages related to factors like technical requirements, costs, reproducibility, and amount of polymorphism detected. The choice of marker depends on the application and study objectives.
Prions are infectious proteins that can replicate by converting normal prion proteins (PrP-sen) into an abnormal disease-causing form (PrP-res). PrP-res accumulates and forms amyloid fibers that are toxic to cells and ultimately cause fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). While prions do not contain genetic material, they propagate by inducing PrP-sen to adopt the abnormal PrP-res conformation. Polymerase chain reaction (PCR) is a technique used to amplify specific DNA sequences, allowing minute amounts of DNA to be analyzed. It involves repeated cycles of heating and cooling of the DNA to separate and copy the strands
This document discusses several neurodegenerative diseases including prion diseases, Alzheimer's disease, and Parkinson's disease. Prion diseases are a group of rare progressive conditions caused by prions, which are abnormal folded proteins. Common types include Creutzfeldt-Jakob disease and variant CJD. Alzheimer's disease is characterized by beta-amyloid plaques and neurofibrillary tangles leading to dementia. Parkinson's disease results from degeneration of dopaminergic neurons and is associated with Lewy bodies containing alpha-synuclein.
This document contains summaries of 4 poster presentations from a conference on Alzheimer's disease and dementia research.
The first poster discusses findings that the ER stress transcription factor XBP1s protects against amyloid-beta neurotoxicity in a fruit fly model of Alzheimer's and in human neuroblastoma cells. XBP1s prevents the accumulation of free calcium in the cytosol, explaining its neuroprotective effects.
The second poster reports that amyloid-beta oligomers impair neuronal function and morphology in rat and mouse neuronal cultures and hippocampal slices through a mechanism linked to NMDA receptor signaling and activation of the protein Jacob.
The third poster examines the role of O-GlcNAc glycosylation in hippocampal synaptic
Presentation made by Dr. Simon Alberti on October 30, 2015 at the Alzforum-hosted live webinar titled "Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?"
More information and the recording of the session available at http://www.alzforum.org/webinars/fluid-business-could-liquid-protein-herald-neurodegeneration
The document discusses DNA replication in prokaryotes and eukaryotes. In prokaryotes, replication begins with initiation at the origin of replication (oriC) mediated by DnaA and other proteins. Bidirectional replication forks are established with the help of helicases and DNA polymerases. Elongation involves continuous synthesis of leading and discontinuous synthesis of lagging strands in Okazaki fragments. Termination occurs when the replication forks from opposite directions converge. In eukaryotes, replication is regulated by cyclin-dependent kinases and occurs at multiple origins of replication with similar initiation and elongation processes as prokaryotes.
The document summarizes 4 projects being conducted as part of a PhD program:
1) Studying the effect of G protein lipids on membrane structure using molecular dynamics simulations. Results show the lipids interact differently with the membrane.
2) Examining 2-hydroxy arachidonic acid as a potential anti-inflammatory drug through binding energy and docking analyses with COX enzymes. It may have advantages over arachidonic acid.
3) Investigating how the drug BGP-15 remodels plasma membrane rafts using density profiles. It enhances docking with cholesterol-rich regions and increases order.
4) Developing GRIMD, a system for distributed molecular dynamics simulations across multiple machines
The document discusses several key concepts in molecular biology including DNA, RNA, transcription, translation, and protein synthesis. It explains that DNA is transcribed into RNA which is then translated into proteins. It describes the central dogma of molecular biology and provides more details on processes like transcription, splicing, and translation. It also discusses topics like alternative splicing, RNA editing, and RNA interference.
This document summarizes Federica Campana's doctoral thesis on investigating drug-cell membrane interactions using molecular dynamics simulations. The thesis examines how membrane composition influences the effects of membrane fluidizers and heat shock protein co-inducers. It also analyzes the binding of anti-inflammatory molecules like hydroxyarachidonic acid to cyclooxygenase enzymes. The overall goal is to better understand how drug molecules interact with and modulate lipid bilayer properties at a molecular level.
Genetics is the study of heredity and genetic variation. Key terms include:
- Genotype is the genetic makeup of an organism, phenotype is observable traits.
- Genes hold information to build cells and pass traits to offspring. The human genome contains 25,000-35,000 genes located on 23 chromosome pairs in the nucleus.
- DNA is transcribed to RNA and translated to proteins, which determine an organism's traits. Variations in genes and chromosomes can result in genetic disorders. Common methods to study genetics include karyotyping, analyzing pedigrees, and identifying alleles and mutations. Understanding genetics provides insight into inheritance patterns and human health.
The genetic code is the system by which nucleotide sequences in mRNA determine the amino acid sequences in proteins. The genetic code uses triplets of nucleotides called codons to specify which amino acid will be incorporated into the growing polypeptide chain. There are 64 possible codons but only 20 standard amino acids, so most amino acids have multiple codons. Three codons act as stop signals to end protein synthesis. The genetic code is nearly universal across all life due to its high degree of specificity and redundancy.
The Drosophila protein Rolling Blackout (RBO) is required for synaptic vesicle endocytosis, as mutants show defects in vesicle recycling and endosome formation; RBO localizes to synapses and interacts genetically with the SNARE protein Syntaxin-1A, suggesting it functions in vesicle fusion or membrane uptake during endocytosis; Loss of RBO specifically impacts endocytosis, as evidenced by accumulation of docked vesicles and defects in dye uptake during stimulation.
This document discusses the learning objectives for a lesson on molecular genetics. It covers DNA structure and function, genes, genetic transfer between organisms, and the effects of genetic engineering on society. Specifically, it aims to explain how the human insulin gene can be inserted into bacterial DNA to produce human insulin through fermentation. It also aims to discuss the social and ethical implications of genetic engineering using a real-world example.
This document describes a study that used CRISPR/Cas9 genome editing to generate zebrafish models of human cardiovascular disorders. The researchers introduced specific point mutations into zebrafish genes linked to dilated cardiomyopathy and Cantu syndrome in humans. They characterized the resulting zebrafish lines and found they exhibited cardiac anomalies and cerebral vasodilation similar to the human conditions. High-speed imaging was used to analyze cardiac function and blood flow in the mutant fish. The study demonstrates that CRISPR/Cas9 can be used to precisely model human genetic diseases in zebrafish at the single nucleotide level.
This document summarizes a study that developed a simplified method for purifying the Thermus aquaticus (Taq) DNA polymerase expressed in Escherichia coli. Key steps included:
1) Overproducing the Taq DNA polymerase in E. coli by cloning the Taq gene into an expression vector and inducing high-level expression.
2) Lysing the E. coli cells and heat-treating the lysate to inactivate endogenous nucleases.
3) Precipitating the Taq polymerase from the lysate using polyethyleneimine and eluting it from an ion exchange resin.
4) Obtaining a purified Taq polymerase preparation in a single-day procedure yielding 40-60 mg of
Structure-Function Analysis of POR MutantsAYang999
This document summarizes protein structure and function, with a focus on mutations in the protein cytochrome P450 oxidoreductase (POR). It describes how mutations can alter a protein's amino acid sequence and structure. Specifically, it investigates the S102P and R550Q mutations in POR, which were found in humans. Experiments expressed and purified these mutant POR proteins, and will test their electron transfer activity compared to the wild type protein using a cytochrome c reduction assay. This will help determine if these mutations impair POR function.
The document describes an experiment where disulfide bonds were engineered into a1-antitrypsin (a1-AT) to lock the movement of helix F (hF) and the connecting loop (thFs3A) during protease inhibition. Disulfide bonds were introduced between thFs3A and strands 3A or 5A of the beta-sheet. The disulfide between thFs3A and strand 5A, but not 3A, eliminated inhibitory activity, suggesting displacement from strand 5A is required for inhibition. The disulfide between thFs3A and strand 3A slowed polymerization without affecting inhibition. This provides direct evidence that hF/thFs3A displacement from strand 5
This presentation aims at giving a vivid knowledge about Nucleoli, a sub organelle of Nucleus, its role in protein formation. control, localization ad how specifically it is involved in Single Nucleotide polymorphism. The slide also discusses about the involvement of SNPs in Alzheimer’s Disease.
Regulation of pten activity by its carboxyl terminal autoinhibitoryChau Chan Lao
Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain.
Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan
From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007
目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白,其由403個氨基酸組成,主要分PTPase及C2 domain,C2 domain使PTEN可與細胞膜作用連結。
PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385),這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株,發現這些變異株具有更強的腫瘤抑制能力,但穩定性將降低,這可能是因這些變異株具有更開放結構所致。
本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株,發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro,而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低,因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法,發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用,暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide,發現其可抑制PTEN之催化活性in vitro,而在細胞表現此peptide則會抑制PTEN之membrane localization,磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。
The genetic code contains rules for translating genetic information in DNA into proteins. It uses triplet codons where each codon specifies a single amino acid. The code is degenerate, with more than one codon coding for an amino acid. It is non-overlapping and non-ambiguous, with each codon corresponding to only one amino acid. Translation proceeds continuously in a 5' to 3' direction, with AUG always initiating protein synthesis and specifying methionine.
This document reports on a study that investigated the effects of oxidative damage to mRNA on translation. The researchers found that a single oxidative lesion, 8-oxoguanine (8-oxoG), drastically reduced the rate of peptide bond formation during translation by more than three orders of magnitude, regardless of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of tRNA selection. This suggests that the modification causes the translational machinery to stall. Consistent with these findings, mRNAs containing 8-oxoG were observed to accumulate and associate with polyribosomes in yeast strains where mRNA surveillance mechanisms were compromised, providing evidence that cells have evolved to cope with damaged mRNA.
The document describes several classes of molecular markers used in genetic analysis, including isozymes, RFLPs, RAPDs, AFLPs, microsatellites, and SNPs. Isozymes analyze differences in protein mobility on a gel, while RFLPs, RAPDs, AFLPs detect DNA fragment length polymorphisms. Microsatellites analyze differences in repeat number, and SNPs detect single nucleotide differences. Each method has advantages and disadvantages related to factors like technical requirements, costs, reproducibility, and amount of polymorphism detected. The choice of marker depends on the application and study objectives.
Prions are infectious proteins that can replicate by converting normal prion proteins (PrP-sen) into an abnormal disease-causing form (PrP-res). PrP-res accumulates and forms amyloid fibers that are toxic to cells and ultimately cause fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). While prions do not contain genetic material, they propagate by inducing PrP-sen to adopt the abnormal PrP-res conformation. Polymerase chain reaction (PCR) is a technique used to amplify specific DNA sequences, allowing minute amounts of DNA to be analyzed. It involves repeated cycles of heating and cooling of the DNA to separate and copy the strands
This document discusses several neurodegenerative diseases including prion diseases, Alzheimer's disease, and Parkinson's disease. Prion diseases are a group of rare progressive conditions caused by prions, which are abnormal folded proteins. Common types include Creutzfeldt-Jakob disease and variant CJD. Alzheimer's disease is characterized by beta-amyloid plaques and neurofibrillary tangles leading to dementia. Parkinson's disease results from degeneration of dopaminergic neurons and is associated with Lewy bodies containing alpha-synuclein.
This is my report on our cell biology. I hope it could help you.
Objectives: Identify infectious proteins (PrPsc), difference of PrPc and PrPsc, list of neurodegenerative diseases that caused by prions.
Prion Diseases ; An overview .........
Credit goes equally to Dr Siraj Uddin, M.V.Sc Scholar, IVRI and Dr. Gazanfar Abass, M.V.Sc. Scholar, VPH, IVRI.
For Further reference contact at gazanfar0966@gmail.com
- Prion diseases are rapidly progressive dementias caused by infectious proteins called prions. They include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), and kuru.
- Prion diseases occur sporadically in 85% of cases, are inherited in 15% of cases, and are infectious in less than 1% of cases. They result from the accumulation of abnormal prion protein (PrPsc) which differs from its normal cellular form (PrPc).
- Clinically, prion diseases present with dementia, myoclonus, and death usually within a year
Prions are proteinaceous infectious particles that lack nucleic acid and cause fatal neurodegenerative diseases. They exist in two forms - a normal cellular form (PrPc) and an abnormal disease-causing form (PrPsc). PrPsc has a different structure than PrPc which allows it to accumulate and convert PrPc into more PrPsc, ultimately causing neuropathology. Prion diseases affect both humans and animals and manifest as infectious, genetic, and sporadic disorders with varied clinical presentations. Common features include neurological deficits, dementia, and spongiform changes in the brain. Prions are extremely resistant to decomposition and differ from bacteria and viruses by being protein-only entities that propagate through conversion of
Prion diseases are rare neurodegenerative disorders caused by misfolded prion proteins. They affect both humans and animals. In cattle it is known as bovine spongiform encephalopathy (BSE) or "mad cow disease", and in humans it is known as Creutzfeldt-Jakob disease (CJD). Prion diseases occur when normal prion proteins misfold and induce other prion proteins to also misfold, triggering a chain reaction that causes damage to neural cells. There is no cure for prion diseases and diagnosis is difficult since prion proteins are similar to normal forms.
Proteins are fundamental components of living cells and come in many forms like hemoglobin, hormones, antibodies, enzymes, and structural proteins. Proteins have a primary structure defined by their amino acid sequence and commonly form secondary structures like alpha helices and beta sheets. For a protein to function properly it must fold into its correct three-dimensional tertiary structure. Misfolded proteins can aggregate and poison cells. Prions are abnormal infectious protein particles that cause neurodegenerative diseases by converting normal cellular prion proteins into the abnormal misfolded prion form.
The document summarizes the structure and effects of prions, which are abnormal protein particles that cause fatal neurodegenerative diseases. Prions have two main forms: PrPc, the normal cellular prion protein consisting of four helices and two beta sheets, and PrPsc, the pathogenic form which has two helices and four beta sheets. PrPsc is highly resistant to heat and enzymes and converts PrPc into more PrPsc via a chain reaction, accumulating in the brain and gradually damaging neurons over time. Different prion strains preferentially affect different brain regions, ultimately leading to sponge-like brain tissue destruction and death from related diseases like Creutzfeldt-Jakob disease in humans and bovine
- In the 1950s, a disease called kuru was observed among the Fore people of New Guinea that caused trembling, loss of motor control, and ultimately death.
- Kuru and other transmissible spongiform encephalopathies (TSEs) are caused by prion proteins, which are unique infectious proteins that can reproduce on their own and convert normal prion proteins into the abnormal infectious form.
- Prions are extremely resistant to heat and chemicals and can survive in the environment for many years. They infect the central nervous system and cause neurodegeneration through a chain reaction that multiplies copies of the infectious prion protein.
Prion diseases are rapidly progressive neurodegenerative disorders caused by abnormal prion proteins (PrP) that can be sporadic, inherited, or transmitted. They include Creutzfeldt-Jakob disease (CJD) in humans and related diseases in other animals. Prion diseases are characterized by brain vacuoles and dementia. Normal PrP converts to an abnormal beta-sheet form (PrPsc) that accumulates in the brain and causes neurodegeneration through an unknown mechanism. Variant CJD results from exposure to bovine spongiform encephalopathy. There are no cures, so prevention focuses on infection control and screening high-risk populations.
The document discusses the history and biology of prions. It covers:
- Key historical prion disease discoveries from the 1730s to 2005.
- The cellular trafficking and structures of normal PrPc and pathogenic PrPsc proteins.
- How PrPsc is able to induce the misfolding of PrPc and replicate.
- Characteristic features of prions like resistance to heat, enzymes, and their ability to spontaneously induce misfolding.
This document presents information on protein folding and mechanisms that conserve protein folding. It discusses how chaperones, such as the GroES/GroEL complex, assist in protein folding. The GroES/GroEL complex uses ATP to encapsulate proteins and release them once folded. Chaperones help prevent protein misfolding and aggregation, which can cause diseases. Clinical examples provided include how protein aggregation can lead to diseases like sickle cell anemia, cystic fibrosis, Huntington's, Alzheimer's, and Parkinson's.
This document summarizes a study investigating the role of a Plasmodium falciparum S33 proline aminopeptidase (PfPAP) in changes to host red blood cell deformability. The key findings are:
1) PfPAP contains a predicted protein export element suggesting it is exported into infected red blood cells. In silico modeling and recombinant protein studies confirmed PfPAP is a proline aminopeptidase.
2) Genetic deletion of PfPAP in P. falciparum led to an increase in the deformability of infected red blood cells and reduced adherence of infected cells to the endothelial cell receptor CD36 under flow conditions.
3) These results suggest PfP
Prion Infection Dynamics- An Analysis of Conversion Mechanisms to Characteriz...Henry Ward Williams III
This document summarizes research on the mechanisms of prion conversion from the healthy cellular prion form (PrPC) to the infectious scrapie prion form (PrPSc). It discusses four main hypotheses: 1) Prusiner's linear autocatalysis model where PrPSc acts as a template to convert PrPC, 2) Prusiner's cooperative autocatalysis model where oligomeric PrPSc lowers the activation energy for additional conversions, 3) the possibility of heterogeneous or homogeneous binding domains between PrPSc and PrPC, and 4) Lansbury's nucleation-dependent model requiring formation of a PrPSc tetramer to accelerate fibrilization. Understanding prion conversion kinetics could provide insights
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
The document reviews the current literature on prion protein (PrP) function and the pathogenesis of prion diseases, focusing on two areas: the unfolded protein response (UPR) and PrP interactions with metal ions. It describes PrP genetics and structure, the UPR pathways involving IRE1, PERK and ATF6, links between the UPR and prions, and the roles of copper and zinc binding in PrP function and prion diseases. The aim is to establish connections between the UPR and PrP metal ion interactions to further our understanding of PrP pathogenesis and native function
Pyruvate kinase (PYK) is a key regulatory enzyme in glycolysis that is allosterically regulated. There are two "secrets of life" - the structure of DNA and allostery. PYK exists in different isoforms and states of activity depending on the presence of effector molecules like fructose-1,6-bisphosphate (F16BP). The M2 isoform found in cancer and embryos is regulated by F16BP and exists in equilibrium between inactive dimers and active tetramers. Understanding PYK regulation may enable new anti-parasitic and anti-cancer drugs by stabilizing different conformational states. X-ray crystallography of Leishmania PYK with and without F26BP
This document discusses genetics and orthodontics. It covers the history of genetics, basic genetic terminology like DNA and genes. It discusses Mendel's laws of inheritance and how DNA is replicated. Part two will cover topics like homeobox genes, twin studies, and the genetics behind malocclusion, tooth agenesis, and other dental issues. Homeobox genes like Msx, Dlx, and Lhx play important roles in craniofacial development and patterning of the dentition. Mutations in these genes can lead to issues like cleft lip and palate.
M Sc Molecular Biology Final- project SV.pptxOmekhan1
Molecular biology is the study of biological molecules and their interactions. The central dogma describes the flow of genetic information from DNA to mRNA to protein. DNA contains the genetic code and is made up of nucleotides with a double helix structure. During transcription, RNA polymerase uses one DNA strand as a template to produce mRNA. The mRNA then directs protein synthesis during translation using ribosomes. Techniques like gel electrophoresis and agarose/polyacrylamide gels are used to separate DNA, RNA, and proteins based on size and charge.
Proteins – Basics you need to know for ProteomicsLionel Wolberger
The document provides an overview of key concepts in proteomics, including:
1) It discusses protein structure and function, the 20 common amino acids, and post-translational modifications that proteins undergo.
2) It introduces common techniques used in proteomics like chromatography, electrophoresis, mass spectrometry, and bioinformatics.
3) It summarizes protein analysis methods like gel electrophoresis, isoelectric focusing, and immunological assays used to detect and purify proteins of interest.
This document provides an overview of protein structure from primary to quaternary levels. It discusses the building blocks of proteins including amino acids and peptide bonds. Secondary structures like alpha helices and beta sheets are explained. Tertiary structure refers to the global folding of the protein chain. Quaternary structure involves the assembly of multiple protein subunits. Examples are given of protein complexes demonstrating tertiary and quaternary levels of structure. The document also outlines different classes of proteins based on function, structure, and cellular localization.
Research report (alternative splicing, protein structure; retinitis pigmentosa)avalgar
This presentation explains the two major scientific projects I have been involved in.
It extends way further than a CV, but shorter than an actual scientific paper.
CRISPR- Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells.- a paper is taken for lab presentation. A very good technique having advantages over conventional KO approaches and allow for the generation of clean CRISPR/ Cas9- based KOs.
DNA and RNA are the two main nucleic acids found in cells. DNA is located in the nucleus and mitochondria where it serves as the genetic blueprint. It has a double-stranded structure and contains the sugar deoxyribose. RNA is single-stranded and found in the nucleus and cytoplasm. It contains the sugar ribose. The three main types of RNA are rRNA, tRNA, and mRNA. rRNA forms the structural framework of ribosomes. tRNA transfers amino acids to the ribosome during protein synthesis. mRNA carries genetic information from DNA to the ribosome to direct protein synthesis.
This study investigated the role of astrocytes and neuronal PrPC in prion-induced neurodegeneration. The researchers set up cocultures of prion-infected astrocytes and uninfected neurons from transgenic mice expressing different PrP variants. They found that interaction between neuronal PrPC and exogenous PrPSc was not sufficient to induce neuronal death, but efficient conversion of neuronal PrPC into PrPSc was required for prion-associated neurotoxicity. Prion-infected astrocytes accelerated neurodegeneration in homologous cocultures compared to infected single neuronal cultures, despite no detectable neurotoxin release. Accumulation of PrPSc in neurons led to neuritic damage and cell death potentiated by glut
The document provides an overview of genomics and proteomics. It defines genomics as the study of an organism's complete set of genes and discusses structural, functional and comparative genomics. It also defines proteomics as the study of the complete set of proteins and discusses structural, functional and expression proteomics. The key techniques discussed for both include sequencing, 2D gel electrophoresis, mass spectrometry and database searching.
This document describes research into the roles of prostaglandin receptors EP2 and EP4 in bone formation. The researchers aimed to clone and express the rat EP2 and EP4 receptors in cells to establish an assay for measuring cyclic AMP levels following ligand binding. Initial colorimetric assays were unsuccessful, but a quantitative enzymatic assay indicated varying transfection success rates depending on DNA-Lipofectamine ratios. Future work will determine optimal transfection conditions and test other prostaglandins to investigate potential anabolic treatments for osteoporosis.
This document discusses genetic polymorphisms and their role in disease pathogenesis and complications. It begins with definitions of health and disease, then discusses various disease causative agents including environmental, genetic, and microbial factors. It describes the organization of the human genome and genes. The document outlines DNA structure and organization, including DNA packaging into chromosomes. It discusses polymorphisms, mutations, and how they can be analyzed using restriction endonucleases and techniques like PCR and gel electrophoresis. Specific examples are provided of how genetic variations can impact disease by changing enzymes, receptors, transport proteins, and other molecular factors.
The researchers aimed to sequence and analyze glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Cyperus alternifolius and Schefflera actinophylla, two plants endemic to Puerto Rico. They cloned the GAPDH genes from each plant into E. coli bacteria and determined the DNA sequences. Bioinformatics analysis would allow comparison of the GAPDH sequences between the two plants and with other previously sequenced GAPDH genes, helping to understand evolutionary relationships and conserved protein regions. Preliminary results showed successful cloning and transformation of GAPDH genes from each plant species.
1. Human Prion Protein Amyloid: A Tale of Structure and Stability Nathan J. Cobb Department of Physiology and Biophysics Case Western Reserve University, Cleveland, OH
12. PrPC PrPSc Digestion with Proteinase K 23 231 23 231 S S S S Proteinase K Proteinase K 231 ~87-90 S S Surrogates for Structural Information Binding of ‘Amyloid-Specific’ Dyes FTIR spectra of brain-derived PrP — PrPC - - - PrPSc ••••• PrP 27-30 PrPC PrPSc PK - + - + -39 kDa -28 kDa Staining with Hematoxylin-Eosin -19 kDa -14 kDa Congo Red Thioflavin T Immunostaining for Glial Fibrillary Acidic Protein Immunostaining for PrP Pan et al. (1993) PNAS Aguzzi et al. (2001) Nature
13. Nelson et al. (2005) Nature Amyloid Fibrils X-ray diffraction structure of microcrystals formed by the peptide GNNQQNY Aβ1-40 structure as determined by solid-state NMR Like other neurodegenerative diseases such as Alzheimer’s and Parkinson’s, TSEs are associated with neuronal accumulation of amyloid deposits Petkova et al. (2004) PNAS
14. Conversion of rPrP (residues 90-231) into Amyloid Fibrils: ‘Synthetic Prions’ Nucleation 2% seed No seed Elongation Fragmentation 2o Nucleation
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16. Site-Directed Spin Labeling Involves substitution of cysteinefor native residues followed by thiol-specific modification with the nitroxidereagent Nitroxide-labeled proteins yield three important pieces of information 1) Distance estimates – dipolar broadening and spin exchange 2) Mobility information 3) Accessibility
17. MS = 1/2 Hres Hres Hres D(Absorption)/dH MI = 1/2 Absorption Energy H H H signal amplitude modulation amplitude EPR spectroscopy Absorption of electromagnetic radiation by an unpaired electron in an applied magnetic field Energy d(Absorption)/dH MI = -1/2 Absorption MS = -1/2 H H H
18. +1 MI MS +1/2 0 -1 Energy -1 -1/2 0 +1 H Nitroxide EPR spectra Distance Estimates Mobility Dipolar Broadening Jayasinghe & Langen (2004) J. Biol. Chem Spin Exchange Margittai & Langen (2004) PNAS
19. Initial Purification and Refolding of rPrP Denaturing Wash (10 mM reduced glutathione, 6 M GdnHCl, pH 8.0) Gradient from 6 M -> 0 M GdnHCl Wash Low Imidazole (50 mMimidazole, pH 8.0) Collect High Imidazole (350 - 500 mMimidazole, pH 5.8 - 6.4) Standard Buffer: 100 mM phosphate, 10 mMTris, pH 7.0
25. EPR Signals for Nitroxide-Labeled rPrP Fibrils Cobb et al. (2007) PNAS
26. 2 M GdnHCl 1 M GdnHCl no denaturant Denaturation of Nitroxide-Labeled rPrP Fibrils Monomer Fibril 4 M GdnHCl Fibril (no denaturant) Fibril 4 M GdnHCl Cobb et al. (2007) PNAS
27. 191 undiluted 1:4 dilution 190/191, 191/192 undiluted 191 1:1 dilution 1:1 dilution Only a Parallel In-Register -Structure can Describe EPR Data -helix -sheet
32. 231 90 H/D exchange (170-220) EPR data (160-220) Correlation with H/D Exchange Data Lu et al. (2007) PNAS
33. Left-handed helical model (89-175) Spiral model (116-164) H/D exchange (170-220) EPR data (160-220) Structural Models of Prion Protein Aggregates Pathogenic Mutations of the Prion Protein E196K T188K T188R F198S E200K D202N H187R V203I Insertion of 1,2, or 4-9 repeats R208H T183A V210I 253 P105L P105T Y145stop M232R V180I E211Q P238S Sim and Caughey (2008) Neurobiol. Aging Wille et al. (2002) PNAS Left-handed β-helical model Govaerts et al. (2004) PNAS Parallel an in-register β-structure of rPrP amyloid Cobb et al. (2007) PNAS Q212P P102L A117V D178N Spiral model DeMarco and Daggett (2004) PNAS Q217R Q160stop 1 51 91 23 90 231
34. Similar Amyloid Fibrils are Formed under Native Conditions Native conditions 50 mM acetate pH 4.0 Denaturing Conditions 50 mM phosphate 2M GdnHCl, pH 7.0 Cobb & Surewicz (2008) J. Biol. Chem.
35.
36.
37. Native and Denaturing Conditions Form rPrP Fibrils of Similar Stability Against pH Against GdnHCl ● Denaturing ○ Native ●EPR ● Denaturing ○ Native ●EPR Monomer Fibril 4 M GdnHCl Cobb & Surewicz (2008) J. Biol. Chem.
38. 179 179 214 214 pH 4.0-10.0 Highly Acidic Conditions Stabilize rPrP Amyloid Fibrils Fewer Stacked Charges at Very Low pH rPrPAmyloid is more Resistant to GdnHCl at Low pH pH 2.0
43. Acknowledgements Witold K. Surewicz Frank D. Sönnichsen HassaneMchaourab Adrian C. Apetri Xiaojun Lu This work was funded by NIH grants NS 44158, NS 38604, NS 14359 (to W.K.S.), and NIH Training Grant T32 HL07653 (to N.J.C)
First of all I would like to thank everyone here at NGM for inviting me and coming to listen to my talk. Our lab at Case Western is interested in pursuing a really muti-diciplinary approach to understanding both structural and mechanistic aspects of the conformational conversion of the prion protein. As you can see by the title of my talk, my postdoctoral work has focused on the structure of amyloid fibrils formed by the recombinant human prion protein.
Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative disorders. These disorders are spread over several mammalian species including CJD, GSS and kuru in humans and most notorioulsy in animals, bovine spongiform encephalopathy or ‘mad cow disease’ in cattle and chronic wasting disease in deer and elk It is the consumption of BSE tainted beef by humans which is believed responsible for the outbreak of vCJD in Britain around the turn of this centuryThese disorders may arise spontaneously as in most cases of CJD, may be inherited as with GSS as well as a variety of CJD-related familial point mutations which predispose an individual to disease, or by infection as is the case with animal TSEs and kuru, arising from the ritualistic cannibalism of brains by the Fore tribe of New Guinea
This protein is expressed mainly in the CNS and is well conserved over all mammalian speciesThe function of PrPC is still unknown, and has been speculated to be involved in copper homeostasis, cell signaling, cell adhesion, anti-apoptotic, protection against oxidative stress, or modulation of synaptic structure and function
In humans, the PrP gene transcribes a 254 amino acid protein containing an N-terminal leader sequence which directs the polypeptide to the cell surface and a C-terminal signal sequence which directs that the attachment of a GPI membrane anchor. Both of these signaling sequences are removed in the mature PrPC resulting which may also be glycosylated at C-terminal asparagines 181 and 197. Here is the solution structure of monomericPrP as determined by NMR using recombinant material. The mature protein consists of an unstructured N-terminal domain which contains the so-called octarepeat region which is able to bind divalent copper, and a globular C-terminal domain which as two short B-sheets, three A-helices and a single disulfide bond between cysteines 179 and 214
In the absence of high resolution structural data for the PrPSc agent, we have relied on surrogate data to reveal that conformational conversion of PrPC to PrPSc is closely linked with the TSE diseases.So here we have a normal cow expressing PrPC which exists in its normal cellular form… so what happens in the ‘mad cow’? **click**First brain histology reveals extensive spongiform degeneration, or vacuolarlization, astogliosis as astrocytes are recruited to phagocytoses dying neurons, as well as accumulation of prion protein deposits **click**PrP aggregates also bind the reasonably amyloid specific dyes Congo Red, which results in so-called apple-green birefringence, and Thioflavin T which can be monitored fluormetrically as is the most common way to evaluate the conversion process in vitro. **click**One commonly exploited biochemical difference between PrPC and PrPSc is the resistance of the latter to proteolytic digestion. So in the presence of the non-specific protease Proteinase K, PrPC is completely digested while PrPSc is cleaved with a ragged end at around sites 87-90 and results in an ~140 amino acid protected region **click**Here we see FTIR spectra of hamster PrP. The solid line is normal monomericPrPC and the amide I band here at about 1650 wavenumbers is characteristic of a primarily A-helical protein, while spectra for either PrPSc or Proteinase K digested PrPSc… this PrP27-30… show a red-shifting to 1630, 1640 wavenumbers which is more characteristic of B-sheet…
I should also mention the difference between infectivity and toxicity of PrP aggregates in the TSE diseases. It now pretty commonly thought that the infectious and neurotoxic agents are distinct species… as with protein misfolding disorders such as Alzheimer’s the neurotoxic species is proposed to be a some smaller, more structurally fluid oligomer… however, with regards to this talk we will be focusing on the infectious PrPSc and not any putative neurotoxic structural speciesX ray diffraction studies of amyloid fibrils have long since been known to reveal a so called cross-B pattern where individual B-strands lie perpendicular to the long fibril axis.In more recent years we have gotten a higher resolution picture of at least some amyloid folds…in this case solid-state NMR has revealed the fibrillar fold of amyloid-beta fibrils associated with alzheimer’s disease **click**And most recently davideisenberg’s group has been able to get X-ray structures of microcrystals formed by short amyloidogenic peptides… Here is the first such structure formed by a peptide isolated from the yeast prion Sup35. In this case B-strands are stacked in parallel and in-register so that same residues are directly above and below same residues on neighboring molecules. Eisenberg’s group has since discovered a range of packing geometries for these short peptide crystals which can be anti-parallel and out-of-register – however all show this so-called steric zipper which is the anhydrous interface between two B-sheets with tight interdigtation of side chains
In my work I am dealing with the in vitro conversion of prion protein to amyloid fibrils. The mechanism by which this occurs is best described by a nucleated polymerization model similar to the formation of crystals. Here we initially observe a lag phase which involves the formation of a stable nucleus, presumably after oligomerization of psome partially unfolded species which is thermodynamically disfavored. After this nucleus is formed, however, it is able to rapidly convert monomeric protein to the amyloid state. If these stable nuclei are added at the beginning of the reaction the lag phase is abolished. Unfortunately, detailed kinetic studies of amyloid fibril growth are far from trivial due to other events which may generate nuclei such as fragmentation of long fibrils or secondary nucleation events, both of which generate additional loci for template-mediated conformational conversion
I should state that the experiments I am about to describe were all performed using the PrP fragment 90-231 which reflects the protease-resistant fragment of PrPSc, contains nearly all predisposing point mutations, and is sufficient for disease. Amyloid fibrils formed from this PrP fragment were shown by Stanley Prusiner’s group to cause disease in mice overexpressing this same construct leading them to conclude that these fibrils represent synthetic prions. However, there are some problems with these studies, rPrPamyloid displays a much shorter PrP resistant core region than authentic PrPSc… more importantly, the infectivity titre of the amyloid fibrils is very low, and these transgenic mice also develop the disease later in life…while the infectivity of the fibrils is dubious at best, they do allow us to examine the conformational space of the prion protein with methods that are not amenable to cellular PrP.
EPR is really just NMR for unpaired electrons which behave like tiny bar magnets in a strong magnetic field. Where can you find unpaired electrons? Examples include free radicals, particular oxidation states of transition metals, and for the present work, the stable unpaired electron of a nitroxide. Of course an electron is much smaller than a proton which is why an EPR instrument needs a magnet which takes up a corner of the room as opposed to filling a room like a NMR instrumentA magnetic field splits Ms=1/2 electron spin states into two energy levels, because of the differences in masses, a given field will split the electron states ~2000 fold more than proton states.After aligning the electron spin with the applied magnetic field we can excite the electron spin into a resonance condition between the electron spin aligned and opposed spin states with microwave radiation. One should see a nice absorption peak at the resonant frequency but because of technical reasons it is easier to vary the magnetic field than the microwave frequency… also, to improve signal to noise the magnetic field is modulated so that noise is filtered out… this results in the measurement not of absolute absorbance but the slope of the absorbance envelope resulting in a derivative spectrum.
In the case of a Nitroxide, the EPR signal is split into a triplet because of the spin 1 Nitrogen nucleus where the unpaired electron residesVery quickly, anytime you see spectra overlaid onto one another, these represent the same molar concentration of nitroxide spins, so it is the overall shape of the line that becomes important – we scale these by normalizing the double intergrals of these spectra to one another, so we have the same area under the curve so to speak, so that broader EPR spectra will display lower amplitude First, these signals give us a site-specific measure of the flexibility of the backbone. This highly isotropic, three line signal here in black would represent a highly mobile backbone, while these broader blue and red signals would indicate increasingly imobilized backbone regions. I should mention that there is also a rotational component to the EPR signal so that a label attached to a small monomeric protein that is tumbling rapidly in solution will show a higher amplitude, more isotropic signal than one that is attached to a protein in a large aggregate such as an amyloid fibrilNext is that you can approximate the distance between spin labels up to about 25 A using conventional EPR… proximity of two labels will result in dipolar broadening of the EPR signal somewhat reminicent of the broadening observed with increasing backbone immobilization… by deconvoluting these signals with a broadening function one can get distance informationThe second proximity regime occurs only when multiple nitroxides are within van derwaal distance of one another and this results in spin-exchange and the emergence of this really striking broad single-line spectrum – experimentally, the only time you ever really can observe these signals are in highly concentrated solutions of nitroxide, crystals of nitroxide… or in certain spin labeled amyloid system
Seeing as how I am interviewing for a position as a protein purification scientist, I want to briefly take you through the rPrPpurifcation protocol. We express an N-terminally His-tagged protein in bacteria using the PET vectors. Given the tendency for prion protein to aggregate, we unsuprizinglyfing it in inclusion bodies which we solubilize in our standard buffer containing 6 M GdnHCl and 10 mM glutathione to reduce and disulfide pairings. We load this onto a Ni column and perform the following purification and refolding scheme on a GE AKTA FPLC. First we have a wash in this denaturing solution followed by a gradient whereby we slowly leech out the denaturant and glutathione to properly refold the protein. We wash and apply 50 mMimidazole to further clean up the column and then we elute the protein - for whatever reason I found that somewhat lower concentrations of imidazole in this elute step really increase the final yield for my cysteine variants. Finally we repeat this whole process minus the low imidazole wash – protein from both fractions are identical the splt in yield is generally around 60/40
For thecysteine mutants, after elution we dialyze them in acetate pH 4 which is where the protein is really happiest and also prevents any intermolecular disulfide formation between the unlabeled cysteines.
Very briefly for any biologist out there I would like to touch upon the potential mechanism of PrPScneuroinvasion, although these processes are still highly controvesialAfter ingestion of the scrapie agent, it may be taken up from the intestinal lumen by microfold cells (i), direct capture by migratory dendritic cells (iii), or by regular intestinal epithelial cells in a complex with ferritin. After this initial uptake, it appears that phagocytosis by macrophages degrades the scrapie agent and are protective, while dendritic cells may transport PrPSc to follicular dendritic cells located in the B-cell rich follicles of peyers patches and gut associated lymphoid tissue. FDCs express PrPC at a relatively high level and may represent the initial location of PrPSc amplification. After incubation in lymphoid tissue in the gut and spleen, PrPSc is presumably able to invade the CNS via enteric nervous system in a process that remains unclear.Then PrPSc invasion of the nervous system occurs in the retrograde direction via sympathetic and parasympathetic nervesIt has been proposed that this retrograde invasion occurs by step-wise interactions of PrPSc with PrPC along the cell surface (i), by fragmentation of large extracellular aggregates, or by some vessicle mediated mechanism (iii). Given that PrPSc spread appears to be directed in the retrograde as opposed to moving to the periphery of the CNS would seem to suggest someform of active transport making this latter mechanism the most likely