Seminar Presentation - Tk subtilisin
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Transcript of Seminar Presentation - Tk subtilisin
Proteolysis of a protease-resistant, infectious
Scrapie Prion Protein using a Thermal Stable Serine
ProteasePresented by:
Alexander Ward
Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1
Authors: Yuichi Koga & Shun-ichi Tanaka & Akikazu Sakudo & Minoru Tobiume & Mutsuo Aranishi & Azumi Hirata & Kazufumi Takano & Kazuyoshi Ikuta & Shigenori Kanaya
Discovery of Prion Disease (PrPSc) Neurodegenerative,
progressive, fatal Unknown origin No isolated bacteria or
viruses? Spongiform neural tissues Types –
Infectious Variant Genetic
"Pathology & Laboratory Medicine." Tulane University. Tulane University School of Medicine, n.d. Web.
Infectious PrPSC
Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.
Types of Prion Diseases (PrPSc)
Prusiner, Stanley B., Michael R. Scott, Stephen J. DeArmond, and Fred E. Cohen. "Prion Protein Biology." Cell, Volume 93, Issue 3, 1 May 1998. Web.
Disease Host Mechanism of Pathogenesis
Kuru Fore people Infection through ritualistic cannibalism
varient Creutzfeldt-Jakob Disease
Humans Infection from bovine prions?
Scrapie Sheep Infection in genetically susceptible sheep
Bovine Spongiform Encephalopathy
Cattle Infection with prion-contaminated Meat and Bone Meal
Chronic Wasting Disease Mule deer, elk Unknown
Feline Spongiform Encephalopathy
Cats Infection with prion-contaminated beef
What is a Prion Protein? (PrPC)
Membrane protein Found in mammalian brain,
spinal and eye tissue 3 CTD α-helices, 2 linked by
a disulfide bond Unstructured NTD NTD has a high affinity for
divalent cations
Baral, P. K., M. Swayampakula, and M.N.G. James. "4MA7." RCSB PDB. PubMed, 22 Jan. 2014.Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.
What is a Prion Protein? (PrPC)
Associated with Immunoregulation Signal transduction Copper binding Synaptic transmission Induction of apoptosis Protection against apoptotic
stimuli
Baral, P. K., M. Swayampakula, and M.N.G. James. "4MA7." RCSB PDB. PubMed, 22 Jan. 2014.Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.
What’s the difference between PrPC and PrPSc?
Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.
a – β-helical model
b – β-spiral model
c – β-sheet model
Amyloid state doesn’t equal neurodegeneration
Decontamination Procedure - WHO
Incineration Autoclave/chemical
methods for dry goods Autoclave/chemical
methods for heat-resistant instruments
Chemical methods for surfaces or heat sensitive instruments
"WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO Consultation, Geneva, Switzerland, 23-26 March 1999." WHO. N.p., Mar. 1999.
Organism – Thermococcus kodakarensis KOD1 (Tk)
Hyperthermophilic Archaea Temp. 60-100o C (opt. 85o C) pH 5-9 (opt. 6.5) Salt conc. 1-5% (opt. 3%) Strictly anaerobic
"Thermococcus Kodakaraensis Genome Project." Imanaka Laboratory, Graduate School of Engineering, Kyoto University, Japan, 17 Feb. 2005. Web. 11 Nov. 2015.
Tk-subtilisin
Tanaka, S., H. Matsumura, Y. Koga, K. Takano, and S. Kanaya. "3A3P." RCSB PDB. PubMed, 22 Oct. 2010. Web. 10 Nov. 2015.
Stable in high temperature, denaturants, and surfactants
7 binding sites for Ca2+
Subtilisin is a serine protease
Serine Proteases
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
Serine Proteases: Hydrolytic Mechanism
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
His 57 acts as a general base, deprotonating Ser 195
Ser 195 nucleophilic attack the carbonyl C of the polypeptide backbone
Serine Proteases: Hydrolytic Mechanism
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
Oxyanion collapses back to form a double bond between the original carbonyl C
His 57 acts as a general acid to protonate the amine leaving group
Serine Proteases: Hydrolytic Mechanism
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
H2O regenerates the active site How to fit the substrate in the
active site?
Denaturation of Proteins
Protonation or deprotonation
Anion or cation interaction
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov. 2015.
Denaturation of Proteins
Interruption through stronger hydrogen bonding molecules
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov. 2015.
Denaturation of Proteins
Reduction of disulfide bonds
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov. 2015.
Denaturation of Proteins
Detergents Interact with non-polar amino
acid residues Small polar molecules
Depends on structure and amino acid composition
Heat can also denature proteins
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov. 2015.
Methods - Enzyme Activity Assay
Azocasein – Measures enzymatic activity using azocasein as a substrate Consists of casein conjugated to an azo-dye
N-succinyl-AAPF-p-nitroanilide (Sigma) Used to test enzyme activity in protein denaturing conditions Cleavage of 4-nitroanilide yields 4-nitroaniline, yellow under basic
conditions
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
Results - Stability of Tk-subtilisin against Denaturants
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
SDS 8M Urea 6M GdnHCl● - Tk-subtilisin∆ - proteinase K□ - Subtilisin Carlsberg
Results - Impact of Surfactants on Tk-subtilisin
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
0.1%1%
Methods - Western Blot
"Western Blot." PPT PowerPoint Drawing Diagrams, Templates, Images, Slides. Motifolio, n.d. Web. 11 Nov. 2015.
Western Blot Analysis
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
1 and 1’ – PrPSc
2 and 2’ – PrPSc and Proteinase K
3 and 3’ – PrPSc and Tk-subtilisin
4 and 4’ – PrPSc, Tk-subtilisin and Proteinase K
Western Blot Analysis
1 and 1’ – PrPSc
2 and 2’ – PrPSc and DFP 3 and 3’ – PrPSc and Tk-
subtilisin 4 and 4’ – PrPSc, Tk-
subtilisin and DFP
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
Western Blot Analysis
A concentration of 0.002mg/mL is enough to digest 60µg of PrPSc in the sample in 3% SDS, at 100o C for 10 minutes.
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
Western Blot Analysis
Two non-ionic industrial surfactants, Emulgen PP290 and 147
Emulsifiers Incubated at 100oC for 10
minutes
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
Western Blot Analysis
sCJD brain homogenate
Incubated at 100oC for 10 minutes
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
Conclusion
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
Special Thanks
Dr. Mary Karpen GVSU Chemistry Department Authors of the article
Questions
Additional Information
DFP – Diisopropyl fluorophosphate – Inhibits some proteases. (C6H14FO3P)
Promazine – Phenothiazine, a class of antipsychotics
DFP to Inactivate Serine Proteases
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
Issues with Decontamination Procedures - WHO It is recommended to discard instruments used in surgery of brain,
spinal cord or eyes of patients with a family history of PrPSc. The size of the macerate and thermostability of the PrPSc, increasing
porous load autoclaving temperature or time is not a reliable decontamination standard.
It has been reported that PrPSc agents are more resistant to inactivation of autoclaving when infected brain-tissue becomes dried onto glass or metal surfaces.
Macerated samples were more resistant to porous load autoclaving compared to intact tissues.
Taylor, D.m. "Inactivation of Prions by Physical and Chemical Means."Journal of Hospital Infection 43 (1999): S69-S76.
Table of Industrial Surfactants used in Tk-subtilisin activity measurements
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2 (2015): 2113-20.
Decontamination Procedure - WHO
"WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO Consultation, Geneva, Switzerland, 23-26 March 1999." WHO. N.p., Mar. 1999.
Chemical disinfectants variably effective
Gaseous disinfectants ineffective
Physical processes variably effective
Urea (6M) Ethylene Oxide Autoclaving at 121oC for 15 minutes
SDS (5%) Formaldehyde Boiling in 3% SDSAlcohol Ionizing UV or
microwave radiation