PROTEIN FOLDING, ASSEMBLY, AND AGGREGATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0219470
• Project No.: NC02318
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2014

Project Director
Clark, A.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Biochemistry

Non Technical Summary
Bovine spongiform encephalopathy (BSE) is linked to the human disease variant Creutzfeldt-Jakob disease (vCJD), and the cost of BSE to European Union member states is estimated at more than 90 billion euros. Because the US beef industry is a $76 billion industry (in 2008), the effects of BSE are potentially devastating to the industry as well as potential health risks to humans. Work in our laboratory has the potential to affect therapeutic strategies for a number of protein aggregation diseases, from Alzheimer?s to Creutzfeldt-Jakob disease to bovine spongiform encephalopathy, because the dysregulation of protein aggregation is a common factor to these diseases. The identification of small molecules that bind to the interfaces of the protein oligomers, resulting in their dissolution is at the forefront of our research.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
723	7010	1000	100%

Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

protein folding

biochemistry

biophysics

protein aggregation

bovine spongiform encephalopathy

alzheimer's disease

Goals / Objectives
Studies of protein folding have had a major impact on the understanding of protein function and the control of human and animal disease. Not only have a number of diseases been attributed to protein folding defects, but the biophysical characterizations of the folding properties suggest therapeutic strategies for treatment. For example, the deltaF508 mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to be a functional chloride channel, but structural alterations prevent its release from the ER. Thus, developing ways to facilitate removal from the ER has important clinical applications in the treatment of cystic fibrosis. Research into the thermodynamic and kinetic properties of the transition of prion protein leads to an understanding of the various forms of transmissible spongiform encephalopathies (TSEs) and can be generalized to other protein aggregation and deposition disorders such as Alzheimer's disease, systemic amyloidosis, and type II diabetes. The pathogenic and infectious form of prion protein, PrPSC, is able to aggregate and form amyloid fibrils, which are very stable and resistant to most disinfecting processes and common proteases. Bovine spongiform encephalopathy (BSE) is linked to the human disease variant Creutzfeldt-Jakob disease (vCJD), and the cost of BSE to European Union member states is estimated at more than 90 billion euros. Because the US beef industry is a $76 billion industry (in 2008), the effects of BSE are potentially devastating to the industry as well as potential health risks to humans.

Project Methods
Under specific conditions, PrPSC in BSE brain tissue was found degradable by a bacterial keratinase and some other proteases. Since the disease-causing prion is infectious and dangerous to work with, model or surrogate proteins are examined in this project that are safe. We have examined three proteins in our studies: yeast Sup35NM, Apaf-1 CARD, and procaspase-3. The three proteins assemble into higher order structures that can be used for modeling protein assembly. Methods used include fluorescence and circular dichroism spectroscopies, x-ray crystallography, and cell biological assays for protein aggregation and activity. Thermodynamic and kinetic studies are used to examine assembly and aggregation pathways for the proteins

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two students graduated with PhD How have the results been disseminated to communities of interest? Through publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Continue to examine chaperone function and native state assembly. Continue to examine allosteric control of activity.

Impacts
What was accomplished under these goals? Several papers published. Investigating new area of intramolecule chaperone activity in forming the protein native state. Also investigating allosteric control of activity.

Publications

• Type: Book Chapters Status: Accepted Year Published: 2014 Citation: 42. Cade, Christine & A. Clay Clark (2014) Caspases, in Proteases in Cell Death, Bose K. (ed.), Springer, in press [Invited Review].
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Ma, Chunxiao, Sarah H. MacKenzie & A. Clay Clark (2014) Redesigning the procaspase-8 dimer interface for improved dimerization
• Type: Journal Articles Status: Published Year Published: 2013 Citation: MacKenzie, Sarah H., Joshua L. Schipper, Melvin Thomas III, Kevin Blackburn, Paul Swartz & A. Clay Clark (2013) Lengthening the intersubunit linker of procaspase-3 leads to constitutive activation, Biochemistry 52, 6219-6231. [PMID: 23941397]
• Type: Journal Articles Status: Published Year Published: 2013 Citation: MacKenzie, Sarah. H. & A. Clay Clark (2013) Slow folding and assembly of a procaspase-3 interface variant. Biochemistry 52, 3415-3427. [PMID: 23614869

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: The long term objective of the lab is to determine the mechanisms that lead to the activation of procaspases, which play central roles in the regulation of apoptosis and inflammation. It has been established that some procaspases, such as procaspase-8, exist as stable monomers while others, such as procaspase-3, exist as stable dimers. Furthermore, dimerization is a key event in maturation of the monomeric procaspases, whereas chain cleavage and subsequent conformational changes are key events in maturation of the dimeric procaspases. Understanding the caspase activation process provides a key to develop strategies to intervene in their activities in the cell. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: We have begun to examine activation of caspases in cell derived from several types of cancer. The goal is to understand how effectively the activated caspases kill cells.

Impacts
Certain human diseases, such as cancer, may be caused by inactive caspases. In other cases, autoimmune diseases may result from overly active caspases. By understanding how caspase-1 and caspase-3 are formed and activated, it may be possible to modify the activation. Without the caspase-1 activator, for example, interleukin-1b would remain inactive thereby preventing the toxic effects on the pancreatic B-cells that result in diabetes.

Publications

• Walters, J., Joshua L. Schipper, Paul Swartz, Carla Mattos and A. Clay Clark (2012). Allosteric modulation of caspases through mutagenesis. Bioscience Reports 32, 401-411.
• MacKenzie, Sarah H. & A. Clay Clark (2012) Death by caspase dimerization, in Protein Dimerization (and Oligomerization) in Biology, J. Matthews, ed., Landes Bioscience, 55-73.
• Schipper, Joshua L., Sarah H. MacKenzie, Anil Sharma & A. Clay Clark (2011) A bifunctional allosteric site in the dimer interface of procaspase-3. Biophysical Chemistry 159, 100-109.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: The long term objective of the lab is to determine the mechanisms that lead to the activation of procaspase-3 and procaspase-8, which play central roles in the regulation of programmed cell death, or apoptosis. It has been established that some procaspases, such as procaspase-8, exist as stable monomers while others, such as procaspase-3, exist as stable dimers. Furthermore, dimerization is a key event in maturation of the monomeric procaspases, whereas cleavage and subsequent conformational changes are key events in maturation of the dimeric procaspases. Understanding the caspase activation process provides a key to develop strategies to intervene in their activities in the cell. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Certain human diseases, such as cancer, may be caused by inactive caspases. In other cases, autoimmune diseases may result from overly active caspases. By understanding how caspase-3 and caspase-8 are formed and activated, it may be possible to modify the activation. Activation of either protein in cancer cells, for example, may induce the cell to undergo apoptosis. The caspase system should be amenable to small molecule drug design for inducing apoptosis in cancer cells.

Publications

• Walters, J., Cristina Pop, Fiona L. Scott, Marcin Drag, Paul Swartz, Carla Mattos, Guy S. Salvesen, and A. Clay Clark (2009) A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis. Biochemical Journal 424, 335-345.
• MacKenzie, S. H., Joshua L. Schipper & A. Clay Clark (2010) The potential for caspases in drug discovery. Current Opinion in Drug Discovery and Development 13, 568-576.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The long term objective of the lab is to determine the mechanisms that lead to activation of procaspase-3 and procaspase-8, which play central rols in the regulation of programmed cell death, or apoptosis. It has been established that some procaspases, such as procaspase-8, exist as stable monomers while others, such as procaspase-3, exist as stable dimers. Furthermore, dimerization is a key event in maturation of the monomeric procaspases, whereas cleavage and subsequent conformational changes are key events in maturation of the dimeric procaspases. Understanding the caspase activation process provides a key to develop strategies to intervene in their activities in the cell. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Certain human diseases, such as cancer, may be caused by inactive caspases. In other cases, autoimmune diseases may result from overly active caspases. By understanding how caspase-3 and caspase-8 are formed and activated, it may be possible to modify the activation. Activation of either protein in cancer cells, for example, may induce the cell to undergo apoptosis.

Publications

• Walters, Jad, Sara L. Milam, and A. Clay Clark (2009)Practical Approaches to Protein Folding and Assembly: Spectroscopic Strategies in Thermodynamics and Kinetics. Methods in Enzymology 455, 1-39.