ORGANIZATION AND REGULATION OF HUMAN PYRUVATE DEHYDROGENASE COMPLEX

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0207332
• Project Start Date: Oct 1, 2006
• Project End Date: Sep 30, 2011
• Program Code: [(N/A)]- (N/A)

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
BIOCHEMISTRY

Non Technical Summary
The research addresses how the pyruvate dehydrogenase complex (PDC) is regulated. PDC plays the determining role as to whether there is net consumption of body carbohydrate reserves. Insulin-resistant diabetics are a very large population, particularly among older adults. In unregulated insulin-resistant diabetes, PDC activity is shut down even when carbohydrate (stored and in the blood) is abundant. Our recent research is providing new insights into the regulation of PDC that, if better understood at the molecular level, may open up new targets for drug development. The long term potential is that this information may support the development of new drugs that can be used to aid the quality of life of adults that are insulin-resistant diabetics.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3899	1000	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3899 - Other animals, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

pyruvate dehydrogenase complex

diabetes

insulin resistant diabetes

pyruvate dehydrogenase kinase

pyruvate dehydrogenase phosphatase

Goals / Objectives
Project 1. Regulation of cardiac PDC by PDP1. We will: a.) support the completion of ongoing studies to determine 3-D structure of PDP1c by preparing SeMet-PDP1c and other preparations. b.) use site-directed mutagenesis to characterize the structural basis for forming the PDP1c Ca2+ L2 complex. c.) determine the conditions that modulate binding and identify specific SMP receptor and other proteins that participate in and regulate binding. d.) evaluate and then utilize PDP1r-specific monoclonal antibodies (MAbs) capacity for interference with binding of PDP1 to SMP and characterize the PDP1r-domain specificity of these MAbs. e). assess and use MAbs not interfering with binding to SMP for isolating proteins that bind to PDP1. Project 2. Molecular basis for PDK2 regulation. PDK2 has a high activity in the absence of K+ ion but lacks both inhibitory and stimulatory regulatory responses. We will: a.) evaluate our proposal of K+ binding behind ATP and ADP at the active site. b.) attempt to crystallize PDK2 with bound ligands and K+ ion and characterize by x-ray crystallography. c.) characterize ligand induced changes in Trp383 of the cross arm upon ligand binding. d.) evaluate changes in cross arm maintenance and interactions with the L2 domain. e.) label PDK3 with an intense fluorescence label and use this PDK3 bound to an anchored E2 60mer in single complex fluorescence microscopy in attempts to directly observe hand-over-hand walking by PDK3 dimer on surface of E2. Project 3. EM studies on human PDC. The objectives are: a.) to locate binding of E3BP within E2-E3BP inner core. b.) to locate binding of E3, PDK3 and PDP1 in the complex. Project 4. Analysis of breathing of E2 core. The objectives are: a.) to get refined understanding of the structural changes occurring with the size change from 220 + or - 20 A with the linked expansion on 2 fold-axis and twisting in the trimers. As in 8 A resolution structure for 220 A set of E2 60mers similar high resolution CEM will be conducted on other size classes. b.) to conduct similar studies with one or more substrate/product present. c.) to use atomic force microscopy (AFM) to evaluate rate of larger size breathing fluctuations with the probe continuously sitting above an anchored core; AFM can detect fluctuations as fast as the 0.1 microsecond time range. d.) to evaluate the effects of substrates on breathing rates.

Project Methods
Project 1. Regulation of cardiac PDC by PDP1. We will: a.) support the completion of ongoing studies to determine 3-D structure of PDP1c by preparing SeMet-PDP1c and other preparations. b.) use site-directed mutagenesis to characterize, within PDP1c subunit structure, the structural basis for forming the PDP1c/Ca2+/L2 complex. c.) determine the conditions that modulate binding and identify specific SMP receptor and other proteins that participate in and regulate binding using mass spectrometry to identify proteins/small molecules required for PDP1 binding to SMP. d.) evaluate and then utilize PDP1r-specific monoclonal antibodies (MAbs) capacity for interference with binding of PDP1 to SMP and characterize the PDP1r-domain specificity of these MAbs. e). assess and use MAbs not interfering with binding to SMP for isolating proteins that bind to PDP1. Project 2. Molecular basis for PDK2 regulation. PDK2 has a high activity in the absence of K+ ion but lacks both inhibitory and stimulatory regulatory responses. We will: a.) evaluate our proposal of K+ binding behind ATP and ADP at the active site using site-directed mutagenesis, activity, and fluorescence. b.) attempt to crystallize PDK2 with bound ligands and K+ ion and characterize by x-ray crystallography. c.) characterize ligand induced changes in Trp383 of the cross arm upon ligand binding using life time fluorescence depolarization with native, W383F-PDK2 and W371F-PDK2. d.) evaluate changes in cross arm maintenance and interactions with the L2 domain using H/D exchange mass spectrometry as well as AUC studies. e.) label PDK3 with an intense fluorescence label and use this PDK3 bound to an anchored E2 60mer in single complex fluorescence microscopy in attempts to directly observe hand-over-hand walking by PDK3 dimer on surface of E2. Project 3. EM studies on human PDC. The objectives are: a.) to locate binding of E3BP within E2-E3BP inner core. b.) to locate binding of E3, PDK3 and PDP1 in the complex using special constructs and a variety of labeling techniques (component in new constructs, antibodies, gold labels). a. and b. will involve using CEM on 3000-5000 molecules of complex at two angles each and symmetry modeling to locate binding of components and using cryotomography analysis of individual molecules. Our laboratory will make preparations. All EM work will be performed by Hong Zhou and colleagues. Project 4. Analysis of breathing of E2 core. The objectives are: a.) to get refined understanding of the structural changes occurring with the size change from 220 + or - 20 A with the linked expansion on 2 fold-axis and twisting in the trimers. As in 8 A resolution structure for 220 A set of E2 60mers similar high resolution CEM will be conducted on other size classes. b.) to conduct similar studies with one or more substrate/product present. c.) to use atomic force microscopy (AFM) to evaluate rate of larger size breathing fluctuations with the probe continuously sitting above an anchored core; AFM can detect fluctuations as fast as the 0.1 microsecond time range. d.) to evaluate the effects of substrates on breathing rates.

Progress 10/01/06 to 09/30/11

Outputs
OUTPUTS: In all animals including farm animals, regulation of the pyruvate dehydrogenase complex (PDC) plays a central role in the tissue-variable gearing up and shutting down of glucose use as a source either of oxidative energy or as source of carbon for synthesis of storage fat. We have continued our studies on the pyruvate dehydrogenase kinase (PDK) isoforms, PDK2 and PDK3. These regulatory enzymes function in switching off the activity of the pyruvate dehydrogenase complex (PDC). We have conducted studies characterizing effects of drugs on PDK2 by activity. We have characterized the effects the very tight binding compounds, AZD7545 and Nov3r, which bind where the lipoyl domain held lipoyl of E2 bind and appear to mimic the acetyl-dihydrolioyl reactive intermediate that stimulates PDK2 activity. These compounds were shown to inhibit E2-activated function of PDK2 by preventing binding of PDK2 to E2 (PDK2's E1 substrate is also bound to the E2 60mer). However, these compounds were shown to stimulate PDK2 activity in phosphorylating free E1; our data indicate that stimulation results from speeding up dissociation of the reaction product, ADP. We have also characterized inhibition of PDK2 by dichloroacetophenone (DCAP) and determined that this tight binding compound probably binds at the same site as pyruvate but does not share the property of pyruvate of giving more than additive (synergistic) inhibition with ADP of PDK2 activity. We have continued studies of the tight binding of PDK3 to lipoyl domain structures (free L2 monomer, GST-L2 dimer and E2 60mer) and characterized the binding of AZD5745 and Nov3r. This work employs mostly analytical ultracentrifugation studies. These are late stage studies needed to strengthen large manuscripts that are already at an advanced stage of preparation. PARTICIPANTS: At KSU (past year): Thomas E. Roche, PI; David Brooks, Research Assistant (part time); Yasuaki Hiromasa, Research Assistant Professor (part time). TARGET AUDIENCES: Scientists interested in the regulation of PDC and basic metabolism to better understand how PDC regulation works under normal conditions and can be controlled in disease states. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of our studies on PDK enhance understanding of the molecular mechanisms involved in the kinase function and regulation. The studies on the structure of the E2 core of the complex will allow greater understanding of the organization and function of PDC.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The pyruvate dehydrogenase kinase (PDK) isoforms are the highly controlled regulatory enzymes that function in switching off the activity of the pyruvate dehydrogenase complex (PDC). In all animals including farm animals, regulation of PDC plays a central role in the tissue-variable gearing up and shutting down of glucose use as an oxidative energy source or as source of storage fat. Among the four PDK isoforms, our work focuses on PDK2 and PDK3. Our intent was to complete preparations that would allow us to complete manuscripts on PDK2 and PDK3. The work on one manuscript is complete that involves an analysis of differences in the capacity of PDK2 and PDK3 to phosphorylate sites 1, 2, and 3 in the E1 component. An interesting aspect is strong evidence that PDK3 moves from phosphorylating site 1 of many E1 to site 2 without PDK3 dimer ever dissociating from the complex. This constitutes evidence for "hand-over-hand" walking of PDK3 while transferring among mobile lipoyl domains. Three other papers are mostly written but each requires additional data. These papers are on very tight binding of PDK3 to mobile lipoyl domains of E2 (using E2 60 mer, free lipoyl domain monomers and GST-held dimers). This work employs mostly analytical ultracentrifugation studies. A second paper on PDK2 mutants focuses on communication from allosteric sites to the active site of PDK2. A third paper involves use of dilute complexes to study PDK2 and PDK3 function. A concerted effort during the summer to make preparations to complete these studies was only partially successful. Another area of research involves collaborative studies using cryoelectron microscopy that focuses on the E2 60mer core, the E2-E3BP subcomplex and inner dodecahedron core of E2. One focus is getting high resolution structure of the inner core, another is establishing how E3BP integrates into the inner core. These studies are with Professor Hong Zhou at UCLA. Samples were prepared and sent to UCLA. PARTICIPANTS: At KSU (past year): Thomas E. Roche, PI; David Brooks, Research Assistant (part time); Mike Aldrete, undergraduate researcher (part time); Yasuaki Hiromasa, Research Assistant Professor (part time). TARGET AUDIENCES: Scientists interested in the regulation of PDC and basic metabolism to better understand how PDC regulation works under normal conditions and can be controlled in disease states. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of our studies on PDK enhance understanding of the molecular mechanisms involved in the kinase function and regulation. The studies on the structure of the E2 core of the complex will allow greater understanding of the organization and function of PDC.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The pyruvate dehydrogenase kinase (PDK) isoforms are the highly controlled regulatory enzymes that function in switching off the activity of the pyruvate dehydrogenase complex (PDC). In all animals including farm animals, regulation of PDC plays a central role in the tissue-variable gearing up and shutting down of glucose use as an oxidative energy source or as source of storage fat. There are four PDK isoforms and targeted inhibition of these enzymes constitutes demonstrated approaches for control of diabetes, providing relief from heart ischemia and killing of cancer cells carrying out Warburg metabolism. Our work focuses on PDK2 and PDK3 isoforms. Our ongoing studies on PDK2 involved the preparation of several new mutants and characterization of these and previously prepared mutants. Our studies on these mutants and wild type PDK2 are providing insights into the molecular mechanisms involved in transmitting regulatory effects from distant regulatory sites to the active site. Beyond understanding fundamental molecular operation, these results will reveal new ways of developing isoform-specific inhibitors. A manuscript is being prepared but there is still some experimental work required to complete these studies. To gain access to its E1 substrate, we have a large body of evidence that establishes that PDK3 dimer moves among lipoyl domains by hand-over-hand transfer between the lipoyl domains of the core-forming E2 60mer. These studies were mostly conducted by Dr. Hiromasa and are nearly completed but some studies are still needed and being conducted as he finds time. He was a postdoctoral in my laboratory for several years and is now employed in our Biotechnology core facility. Succinctly, these papers include one that is a large set studies using analytical ultracentrifuge and an unusual competitive binding approach to demonstrate extraordinarily tight (subnanamolar) binding of PDK3 to two inner lipoyl (L2) domains of the E2. The work establishes much weaker binding by E2's L1 domain but suggest that this domain facilitates faster interlipoyl domain transfer of the PDK3 dimer. A second paper shows that PDK3 sustains high activity using very dilute complexes. A third paper provides evidence that the basis for phosphorylation of a second site on E1 is not due to substrate preference but is due to sustained access to a specific set of E1 bound to an E2 60mer. PARTICIPANTS: At KSU (past year): Thomas E. Roche - PI, David Brooks - Research Assistant (part time), Mike Aldrete - undergraduate researcher (part time), Yasuaki Hiromasa - PD research associate (part time) TARGET AUDIENCES: Scientists interested in the regulation of PDC and basic metabolism to better understand how PDC regulation works under normal conditions and can be controlled in disease states. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of our studies on PDK2 enhance understanding of the molecular mechanisms involved in PDK2 function and regulation. The full set of studies on PDK3 provide strong support for a novel mechanism for this kinase isoform gaining access to its protein substrate. We expect these studies will be submitted and published in strong journals.

Publications

• No publications reported this period

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Studies continue on the regulation of the pyruvate dehydrogenase complex (PDC) and papers are being prepared on our results on pyruvate dehydrogenase kinase (PDK) isoforms. The studies provide new insights into how ligands and the dihydroplipoyl acetyltransferase (E2) component aids and alters PDK phosphorylation of the pyruvate dehydrogenase (E1) component. A paper is under preparation showing new insights into PDK2 regulation. Towards understanding ion effects on PDK2 function, the mechanisms by which Cl− alters PDK2 activity is established using kinetic, ligand binding and PDK2 mutants. Towards understanding how reductive acetylation of lipoyl groups stimulates PDK2 activity, the effects of Nov3r, a tight binding analog of acetyl-dihydrolipoyl group, on the binding of ligands at other sites (regulatory and catalytic) were characterized. For evaluating the hypothesis that PDK3 dimer moves among lipoyl domains to gain access to its E1 substrate by a nondissociative "hand-over-hand" transfer mechanism, a variety of studies are mostly completed. A paper is in preparation that employs analytical ultracentrifugation to evaluate the strength of interactions of PDK3 with a wide variety of lipoyl domain structures. The work establishes very tight bi-functional binding to the inner lipoyl domain of E2 and suggests a role due to much weaker binding to L1 domain fostering faster interlipoyl domain transfer of the PDK3 dimer. Additional studies on this topic of "hand-over-hand" movement characterized how PDK3 operates using very dilute complexes and obtained evidence that the basis for phosphorylation of a second site on E1 is not due to substrate preference but is due to sustained access to a specific set of E1 bound to an E2 60mer that also binds a PDK3. We have also completed extensive studies on kinetic, ligand binding and protein interactions of PDK1 isoform. Dr. Hiromasa who performed these studies has been asked to prepare a manuscript describing these results which demonstrate unique properties of this kinase isoform. PARTICIPANTS: At KSU (past year): Thomas E. Roche, PI; Liangyan Hu, GRA; Yasuaki Hiromasa, PD research associate. TARGET AUDIENCES: Scientists interested in the regulation of PDC and basic metabolism to better understand how PDC regulation works under normal conditions and can be controlled in disease states. PROJECT MODIFICATIONS: Planned studies on pyruvate dehydrogenase phosphatase isoform 1 (PDP1) did not significantly advance due to personnel turnover and limited progress in work attempted.

Impacts
The results of our studies on PDK2 enhance understanding of the molecular mechanisms involved in PDK2 function and regulation. The combined set of studies on PDK3 provide strong support for a novel mechanism for this kinase isoform gaining access to its protein substrate. We expect these studies will be submitted and published in strong journals. The publications (below) that came out during the year are a result of prior studies described in last year's progress report. Since changes in PDK1 function occur in some forms of cancer, the results of our studies on this isoform will have broad interest.

Publications

• Yu X., Hiromasa, Y., Tsen, H., Chiu, W., Stoops, J.K., Roche, T.E., and Zhou, Z.H. (2008) Structures of the human yruvate dehydrogenase complex cores: a highly conserved catalytic center with flexible N-terminal domains. Structure 16, 104-114. With online set of 3 movies.
• Hiromasa, Y. and Roche, T.E. (2008) Critical role of specific ions for ligand-induced changes regulating pyruvate dehydrogenase kinase isoform 2. Biochemistry 47, 2298-2311. With online Supplemental Information.
• Hirmoasa, Y., Yan, X., and Roche, T.E. (2008) Specific ion influences on self-association of pyruvate dehydrogenase kinase isoform 2 (PDHK2) binding of PDHK2 to the L2 lipoyl domain, and effects of the lipoyl group-binding site inhibitor, Nov3r. Biochemistry 47, 2312-2324. With online Supplemental Information.
• Roche, T.E., Peng, T., Hu, L., Hiromasa, Y., Bao, H. and Gong, X. (2008) Role of lipoyl domains in the function and regulation of the mammalian pyruvate dehydrogenase complex in Alpha Lipoic Acid: Energy Production, Antioxidant Activity and Health Effects (M.S. Patel and L. Packer, eds.) Taylor and Francis, Boca Raton, chapter 7, pp 167-195.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: The following studies were on the regulation of the pyruvate dehydrogenase complex (PDC). Research was completed (in press) on how the coupled binding of K+ ion and phosphate (Pi) with ATP, ADP, pyruvate and combinations of these ligands result in critical interbinding site communication that controls the activity of pyruvate dehydrogenase kinase isoform 2 (PDK2). Studies were completed (in press) that demonstrated K+ binding at a second site in PDK2 that fosters PDK2 binding to the inner lipoyl domain (L2) of the E2 (dihydrolipoyl acetyltransferase). Pi was shown to play a critical role in the coupled binding of ADP and pyruvate that hinders to PDK2 binding to L2 and causes PDK2 dimer to associate to a tetramer. Nov3r binding at the site that the lipoyl prosthetic group of L2 binds was shown to alter binding of pyruvate and ADP by a Pi-dependent coupled binding. The importance of developing PDK inhibitors for intervention with diseases (diabetes, heart ischemia, and cancer) was also reviewed. Studies on PDP1 regulation minimally progressed due to turnover of personnel on this project. PDP1c mutants were prepared (need characterization) and we obtained evidence for a modified form of the large regulatory subunit of PDP1r. Further studies will evaluate whether this modification is linked to the sequestering of PDP1 away form PDC in heart mitochondria. Structural studies. Work was complete (in press) on 8 angstrom resolution structure of the inner core of the human E2 component. New insights into unique features in the structure of the mammalian E2 inner core 60mer that differ from bacterial E2 were obtained by homolgy modeling. PARTICIPANTS: At KSU (past year): Thomas E. Roche, PI, Liangyan Hu, GRA, Yasuaki Hiromasa, PD research associate (part time), Varun Kumar Muthu Kumar (GRA, part of year), Danielle Ngaba (research assistant, part of year). Collaboratoras At Univ. of Houston Medical School (past year): James K. Stoops (Professor), Z. Hong Zhou (Professor). TARGET AUDIENCES: Scientists interested in the regulation of PDC and basic metabolism to better understand how PDC regulation works under normal conditions and can be controlled in disease states. PROJECT MODIFICATIONS: None although PDP studies progressed poorly due to personnel turnover.

Impacts
Our work provided important new insights into the regulation of PDK2. This kinase is universally distributed in most body tissues and plays a very important role in determining whether sugar will be used as an oxidative substrate. Our studies explained: 1) how PDK2 activity is inhibited (allowing activation of PDC) due to ion-aided ADP and pyruvate inhibition and prevention of PDK2 binding to PDC (via L2 domain of E2) and 2) how reductively acetylated lipoyl group (mimicked by the high affinity analog Nov3r) transmits signals that stimulate kinase activity to inactivate PDC. Our studies on the structure of the inner core of the complex both provide insights into the fundamental organization of the complex and how E2 subunits are altered (compared to bacterial E2 subunits) to support other roles unique to mammalian E2, including binding of the E3-binding component and holding E1 away from the inner core of E2, so that it is available for the regulatory enzymes to phosphorylate and dephosphorylate.

Publications

• Roche, T. E. and Hiromasa, Y. 2007. Pyruvate dehydrogenase kinase regulatory mechanisms and inhibition in treating diabetes, heart ischemia, and cancer, Cell. Mol. Life Sci. 64, 830-849.
• Roche, T.E., Peng, T., Hu, L., Hiromasa, Y., Bao, H., Gong, X. (2007) Role of lipoyl domains in the function and regulation of the mammalian pyruvate dehydrogenase complex in Alpha Lipoic Acid: Energy Production, Antioxidant Activity and Health Effects (M.S. Patel and L. Packer, eds.) Taylor and Francis, Boca Raton in press.
• Yu, X., Hiromasa, Y., Tsen, H., Stoops, J.K., Roche, T.E., and Zhou, Z.H., (2007 ) Structures of the Human Pyruvate Dehydrogenase Complex Cores: a Highly Conserved Catalytic Center with Flexible N-terminal Domains. Structure in press.
• Hiromasa, Y. and Roche, T.E. (2007) Critical Role of Specific Ions for Ligand-induced Changes Regulating Pyruvate Dehydrogenase Kinase Isoform 2. Biochemistry, in press.
• Hirmoasa, Y., Yan, X., and Roche, T.E. (2007) Specific ion influences on self-association of pyruvate dehydrogenase kinase isoform 2 (PDHK2), binding of PDHK2 to the L2 lipoyl domain, and effects of the lipoyl group-binding site inhibitor, Nov3r. Biochemistry in press.