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


His 57 acts as a general base, deprotonating Ser 195

Ser 195 nucleophilic attack the carbonyl C of the polypeptide backbone



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



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.


Interruption through stronger hydrogen bonding molecules



Reduction of disulfide bonds



Detergents Interact with non-polar amino

acid residues Small polar molecules

Depends on structure and amino acid composition

Heat can also denature proteins


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


SDS 8M Urea 6M GdnHCl● - Tk-subtilisin∆ - proteinase K□ - Subtilisin Carlsberg

Results - Impact of Surfactants on Tk-subtilisin


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


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


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



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.



Two non-ionic industrial surfactants, Emulgen PP290 and 147

Emulsifiers Incubated at 100oC for 10

minutes



sCJD brain homogenate

Incubated at 100oC for 10 minutes


Conclusion


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


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


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

Seminar Presentation - Tk subtilisin

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