Progress 09/01/10 to 08/31/15
Outputs
Target Audience:Research scientists and students in agricultural sciences, biology, and medicine. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student obtained his PhD degree in December 2014. This project provided him an opportunity for obtaining research capability in molecular genetics, cell biology and biochemistry. Moreover, he has published five manuscripts. Two undergraduate students were also participated in the research. How have the results been disseminated to communities of interest?The research outcomes were reported in three manuscripts and presented at the multiple regional and international conferences, including Gordon Research Conference in Cell Biology of Metals, and 12th Asian Congress of Nutrition. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Cadmium is a widely distributed and highly toxic natural mineral. Its levels on earth crust have been elevated by industrial activities and weathering. Cadmium contamination in the diets and smoking are major routes of cadmium intake of humans. Given numerous reports and convincing evidence of implications of cadmium in human diseases, such as damage of organs, in particular liver and kidney, endocrine disruption, and cancer, it's important to define the mechanisms underlying detoxification of cadmium in living organisms. This project has focused on identification a cadmium exporter in baker's yeast as a model organism and characterization of its function and mechanisms of regulation. Our studies would facilitate mechanistic insights into metal detoxification in plants, animals, and humans. This will ultimately promote safe food production and combating diseases that are related to cadmium toxicity. The research outcomes are also significance for biotechnological applications of the cadmium exporter to reduce cadmium contents in plants as a tool for limiting cadmium consumption of humans. (1) Our results indicate that endoplasmic reticulum-associated degradation (ERAD) of pca1p, a polytopic protein occurs via its interaction with the proteasome at the membrane. ERAD plays a critical role for destruction of terminally misfolded proteins at the secretory pathway. The system also regulates expression levels of several proteins such as Pca1p, a cadmium exporter in yeast. Nevertheless, the mechanism by which Pca1p and other polytopic proteins are targeted to the proteasome in the cytosol remains unclear. Our study determined the roles for the molecular factors of ERAD in dislodging Pca1p from the ER. Inactivation of the 20S proteasome leads to accumulation of ubiquitinylated Pca1p predominantly in the ER membrane. This suggests that the proteasome is required for not only destruction but also extraction of Pca1p from the ER. Pca1p formed a complex with the proteasome at the membrane in a Doa10p E3 ligase dependent manner. Cdc48p is required for recruiting the proteasome to Pca1p. While Ufd2p E4 ubiquitin chain extension enzyme is involved in efficient degradation of Pca1p, the defect of Pca1p poly-ubiquitinylation in Ufd2p-deficient cells did not affect formation of a complex between Pca1p and the proteasome. Two other polytopic membrane proteins undergoing ERAD, Ste6*p and Hmg2p, also displayed the same outcomes observed for Pca1p. However, poly-ubiquitinylated Cpy1*p, a luminal ERAD substrate, was detected in the cytosol independent of proteolytic activities of the 20S proteasome. These results indicate that extraction and degradation of polytopic membrane proteins at the ER is a coupled event carried out by the proteasome that is recruited to the ER. This mechanism would relieve the cost of exposed hydrophobic domains in the cytosol during ERAD. (2) We have revealed that cadmium binding and secondary structure of a degradation signal in Pca1p determine its role for expression control of pca1p cadmium exporter. Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast S. cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system in the absence of cadmium via a cis-acting degron. Cadmium induces Pca1p stabilization in a manner dependent on the degron, suggesting cadmium-mediated masking of the signal blocks molecular factors involved in the ERAD of Pca1p. However, the characteristics and mechanisms of action of the degron in Pca1p and most of those in other proteins remain to be determined. Our data presented herein suggests Pca1's degron senses cadmium by specific cysteine residues to induce conformational change. Random mutation of the degron followed by selection of those losing its degron functionality revealed that distribution of amino acid residues and secondary structure forming an amphipathic helix comprise the signal for recruiting molecular factors for degradation. Hydrophobic amino acids and cadmium binding to the degron affect Pca1p's interaction with Ssa1p molecular chaperone which is involved in protein turnover. These results provide new insights into the mechanism of action of a degron.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Smith, N., Adle, D, Zhao, M., Qin, X., Kim, H. and Lee, J. (2015) Endoplasmic reticulum-associated degradation of Pca1p, a polytopic protein, via interaction with the proteasome at the membrane. J. Biol. Chem.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Smith, N., Wei, W., Qin, X., Zhao, M., Seravalli, J., Kim, H. and Lee, J. (2015) Cadmium binding and secondary Structure of a degradation signal, degron, determine its role for cadmium-dependent expression control of Pca1p. J. Biol. Chem.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Zarruk, J.G., Berard, J.L., Dos Santos, R.P., Kroner, A., Lee, J., Arosio, P and David, S. (2015) Expression of iron homeostasis proteins in the spinal cord in Experimental Autoimmune Encephalomyelitis and their implications for iron accumulation. Neurobiol. Dis.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Research scientists in biology and medicine. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A graduate student who has played a major role in conducting the proposed research would obtain his PhD degree in December 2014. How have the results been disseminated to communities of interest? The research outcomes have been reported in two published manuscripts and at a FEASEB summer conference. What do you plan to do during the next reporting period to accomplish the goals? Given the last year of this project, all proposed experiments will be completed in the 2014-2015 reporting period. Two other manuscripts will be published. A graduate student who has worked on the proposed research will be defending his PhD thesis. The PI and co-investigators will develop a new project.
Impacts
What was accomplished under these goals?
Detoxification of non-physiological metals and homeostatic acquisition of nutritional yet toxic metals are fundamental biological processes. Excess exposure to heavy metals in conjunction with environmental contamination or genetic and epigenetic problems in detoxification mechanisms results in serious disorders in all living organisms. Cadmium is a highly toxic metal with no nutritional role and has been implicated in a number of human disorders, including kidney disease, cancer, and endocrine disruption. Given that diet and smoking along with pollution are major sources of cadmium to the general population, cadmium accumulation in agricultural plants largely determines human exposure. Mechanistic insights into cellular cadmium uptake, sequestration and extrusion would facilitate prevention and treatment of cadmium-related disorders and the development of methods for the reduction of cadmium intake in humans. However, the mechanisms of cadmium metabolism, particularly cadmium extrusion to reduce accumulation in human, animals and plant remains to be studied. This project identified a cadmium exporter and has characterized the mechanisms underlying rapid turnover of the transporter in the absence of cadmium and stabilization by cadmium exposure of cells. The transporter directly sense cadmium, which prevents its recognition for degradation. This is an important strategy for organisms to react promptly in response to changes in cadmium levels. Research outcomes defined several cellular factors that are involved in this process. A negative effect of cellular oxidative stress on cadmium detoxification has been ascertained as well. Identification and characterization of a new cadmium extrusion pump and elucidation of the mechanisms by which organisms regulate expression of the transporter allowed us to define a novel cadmium detoxification mechanism and set solid foundation for employing these insights to reduce human exposure. This study would ultimately advance the ability to combat cadmium-related diseases.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Frey, A.G., Nandal, A., Park, J.H., Smith, P.M., Yabe, T., Ryu, M.-S., Ghosh, M.C., Lee, J., Rouault, T.A., Park, M.H., Philpott, C.C. (2014) The iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase. Proc. Natl. Acad. Sci. USA. 111(22):8031-6. (PMCID: PMC4050543)
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Wei, W.,* Smith, N.,* Wu, X.,* Kim, H.,* Seravalli, J., Khalimonchuk, O. and Lee, J. (2014) YCF1-mediated cadmium resistance in yeast is dependent on copper metabolism and antioxidant enzymes. Antioxid. Redox Signal. 21:1475-89. (PMID: 24444374)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Anandhan, A., Rodriguez-Rocha, H., Bohovych, I., Zavala-Flores, L., Rochet, J-C., Lee, J., Khalimonchuk, O. and Franco, R. (2014) Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways. Neurobiol. Dis.
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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 have worked on this project. How have the results been disseminated to communities of interest? Research outcomes were presented at two international conferences. What do you plan to do during the next reporting period to accomplish the goals? We will continue our research projects determining (1) mechanisms of action and regulation of PCA1 cadmium exporter, (2) effects of cadmium on cell signaling, and (3) cadmium-induced perturbation of lipid metabolism.
Impacts
What was accomplished under these goals?
Our research demonstrates that cadmium resistance in yeast is dependent on copper metabolism and antioxidant enzymes. Given the requirement of metallochaperones in cellular copper distribution and metallation of cuproproteins, we investigated whether metallochaperones also deliver metal ions to transporters functioning in metal detoxification. Resistance to excess cadmium and copper of yeast, which is conferred by PCA1 and CaCRP1 metal efflux P-type ATPases, respectively, is independent of known metallochaperones. However, YCF1-mediated cadmium tolerance relies on a copper chaperone, CCS1, for its function in the maturation of Cu/Zn superoxide dismutase as well as another antioxidant enzyme glutathione reductase. This illustrates an intriguing interaction between metal ions and a new role of antioxidant enzymes. YCF1 is a useful model for further studies on redox-dependent regulation of this family of transporters which are associated with several health concerns, such as cystic fibrosis and multi-drug resistance. A manuscript presenting these results has been accepted in a top-tier journal, and follow-up studies are in progress.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ghosh, M.C., Zhang, D-L., Jeong, S.Y., Kovtunovych, G., Ollivierre-Wilson, H., Noguchi, A., Tu, L., Senecal, T., Robinson, G., Crooks, D.R., Tong, W-H., Ramaswamy, K., Singh, A., Graham, B.B., Tuder, R.M., Yu, Z-X., Eckhaus, M., Lee, J., Springer, D.A. and Rouault, T.A. (2013) Deletion of iron regulatory protein 1 causes polycythemia and pulmonary hypertension in mice through translational derepression of HIF2?. Cell Metabolism. 17:271-81. (PMID: 23395173)
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Massilamany, C., Gangaplara, A., Kim, H., Stanford, C., Rathnaiah, G., Steffen, D., Lee, J., and Reddy J. (2013) Copper zinc superoxide dismutase 1-deficient mice show increased susceptibility to experimental autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein 35-55. J. Neuroimmunol. 256:19-27. (PMC3569856)
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The long-term goal of this project is to define cellular metal detoxification mechanisms and employ this knowledge to control metal-induced adverse effects in plants, animals and humans. Studies on the mechanisms that organisms have evolved to defend against metal toxicity have revealed fascinating modes of environment-gene interaction. This project has focused on characterization of the function, mechanisms of action and regulation of Pca1, a cadmium exporting P1B-type ATPase, of baker's yeast Saccharomyces cerevisiae. The central hypothesis is that Pca1 is the first cadmium selective efflux pump at the plasma membrane and is unique in structure, metal specificity and mechanism of regulation. Biochemical, cell biological, genetic and biophysical approaches have elaborated this hypothesis. We also have extended this research for characterization of another cadmium transporter Ycf1, which has revealed novel functional interactions between copper metabolism and cadmium defense. Our research focus of this year was placed on two objectives. (1) Mechanistic understanding of a cadmium-dependent masking of a protein degradation signal that was identified from Pca1. Pca1 N-terminal domain contains an autonomous signal for rapid turnover of Pca1 in the absence of cadmium. We have employed biophysical and genetic approaches to determine how cadmium turns off the degradation signal to promote expression of Pca1 at the plasma membrane. (2) Determination of the role for metallochaperones in resistance to cadmium and copper toxicity. This study ascertained the roles for metallochaperones in metal efflux by P1B-type ATPases. Our results indicate that resistance to excess cadmium and copper conferred by Pca1 and CaCRP1 (Cad1) metal efflux P1B-type ATPases, respectively, does not rely on metallochaperones. Copper deficiency induced by copper extrusion through CaCRP1 (Cad1) also occurs in the absence of Atx1. These results were presented in an international and a regional research conference. One book chapter and one review summarizing research progress in this topic have been accepted for publication. A research manuscript has been submitted to a peer-reviewed journal. PARTICIPANTS: This project provided opportunities for (1) collaboration among four faculty in the Biochemistry Department of University of Nebraska-Lincoln, and (2) training of two postdoctoral fellows and one graduate student. TARGET AUDIENCES: Scientists in the fields of mineral metabolism, post-translational modification of proteins, and regulatory protein turnover would appreciate the research accomplishments of this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Metal ions, both essential nutrients and non-physiological environmental contaminants, are highly toxic when cellular accumulation is in excess or detoxification systems are compromised. Detrimental effects of toxic metals are implicated in various disorders in all organisms. Better understanding of biological effects, metabolic pathways, and cellular responses to metal ion dyshomeostasis would ultimately facilitate development of methods for combatting metal-related disorders, prevention of exposure to plants, animals and humans, and efficient remediation of toxic metals from the polluted environment. Our research progress sheds light on how the cadmium-responding protein degradation signal in a cadmium transporter. This domain manifests one to one binding of cadmium, which is dependent on two di-Cys residues (CXC and CC). Our results suggest the domain is composed of ~50% alpha helices, which is consistent to the predicted structures obtained using softwares. There is very little secondary structure change by cadmium, which indicates that cadmium binding might affects tertiary structure through movement of secondary structural elements. Next, our studies show that cadmium and copper efflux transporters that work primarily for metal detoxification can acquire their substrates without assistance of known metallochaperones. These results challenge the concept of carrier-mediated cellular metal distribution and provide new avenues for elucidating the roles of metallochaperones. Lastly, the specificity of a metallochaperone in efficient copper acquisition rather than direct role for cadmium metabolism is attributed to maintaining functional integrity of a cadmium defense system. This reveals a new implication for normal copper distribution in cadmium defense and an intriguing interaction between metal ions.
Publications
- Smith, N., Wei, W. and Lee, J. 2012. Cadmium transport in eukaryotes. Metals in Cell. In press.
- Kim, H., Wu, X. and Lee, J. 2012. SLC31(CTR) family of copper transporters in health and disease. Mol. Aspects Med. In press.
- Wei, W., Kim, H., Wu, X., Smith, N., Seravalli, J., Khalimonchuk, O. and and Lee, J. 2012. A role for metallochaperones in resistance to cadmium and copper toxicity of yeast Saccharomyces cerevisiae. Submitted.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Cadmium is a highly toxic environmental contaminant, which is implicated in various disorders in all living organisms. Our successful attempt searching for new genes involved in metal resistance in baker's yeast as a model eukaryote identified a novel cadmium exporting P-type ATPase, Pca1. This project has focused on characterization of the function, mechanisms of action and regulation of Pca1. The central hypothesis of this study is that Pca1 is the first cadmium selective efflux pump that is unique in structure, metal specificity, and mode of regulation. This has been tested using biochemical, cell biological, genetic and biophysical approaches. Our research in current year has focused on characterization of a cadmium-responding degradation signal within Pca1(250-350). (1) Biophysical characterization of cadmium binding to a cadmium-responsive degradation signal of Pca1. Pca1(250-350) peptide and the same peptides possessing side-directed mutation of residue(s) responsible for either cadmium sensing or recognition by the ERAD machinery were purified using an E. coli expression system. In vitro studies on cadmium binding affinity and structural characterization of the peptide w/ and w/o cadmium binding or site-directed mutation of predicted cadmium-binding residues have been conducted using biophysical approaches, including UV-fluorescent spectroscopy, Isothermal Titration Calorimetry (ITC), and Circular Dichroism (CD) spectroscopy. Our data indicates that purified Pca1(250-350) binds cadmium specifically, which corresponds to in vivo data. (2) Define the mechanisms underlying cadmium sensing by Pca1(250-350) and cadmium access for transportation via Pca1. Several lines of our data show that Pca1(250-350) directly senses cadmium to enhance expression of Pca1 when yeast cells are exposed to cadmium. An important question is how cadmium is delivered to the cadmium sensing Pca1(250-350) motif. Pca1(250-350) may physically interact with specific cytoplasmic cadmium carrier(s). This hypothesis is based on the fact that copper-transporting P-type ATPases, which are believed to function in a similar manner to cadmium-transporting P-type ATPase Pca1, rely on metallochaperones (metal carrier molecules). Our research progress illustrates that the known cytosolic metallochaperones are not responsible for cadmium delivery to Pca1(250-350) for its cadmium sensing. Moreover, distinct from copper-transporting P-type ATPases, Pca1 cadmium transport function is not dependent on those metallochaperones either. These data suggest that organisms have evolved metallocaherones to cope with metal limitation but not excess. The PI presented the research progress at two conferences; Gordon Research Conference on Metals in Cell Biology, and 14th International Meeting on Trace Elements on Man and Animals. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Scientists in the fields of metal ions and protein turnover would appreciate our research progress. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This research progress provides new insights into the mechanisms of action of Pca1 and other metal-transporting P-type ATPases. Cadmium sensing and transport by Pca1 without assistance of known metallochaperones is an unanticipated but important finding. This data would have broad impact on understanding the functions of metallochaperones and modes of action of metal-transporting P-type ATPases.
Publications
- Jeong, S. Y., Wilson-Ollivierre, H., Ghosh, M.C., Crooks, D.R., Sougrat, R, Lee, J., Cooperman, S., Jortner, B.S., Mitchell, J.B., Beaumont, C. and Rouault, T.A. 2011. Abnormal iron metabolism compromises function and integrity of motor neurons and impairs mitochondrial function in Iron Regulatory Protein null mice. PLoS One. 6(10):e25404.
- Willis, M.N., Liu, Y., Biterova, E.I., Kim, H., Lee, J., and Barycki, J.J. (2011) Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits. Biochemistry. 50:6508-6517.
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Progress 09/01/10 to 11/30/10
Outputs
OUTPUTS: Cadmium is a highly toxic environmental contaminant, which is implicated in various disorders in all living organisms. While cadmium exporters are known to be vital for cadmium defense in bacteria, the counterparts in eukaryotes remain to be studied. The long-term goals of this project are the characterization of molecular mechanisms of cadmium detoxification and employing this knowledge to reduce cadmium exposure to humans. Research objectives focus on characterization of the function, mechanism of action and regulation of Pca1, a cadmium extruding P1B-type ATPase recently identified in yeast Saccharomyces cerevisiae. One of the most intriguing questions in regard to P1B-type ATPase metal transporters is what structural feature and residues in this family of proteins determine the metal selectivity. We employed an unbiased approach to identify all residues and domains contributing to substrate specificity of Pca1. Pca1 random mutant libraries were constructed and expressed in a copper sensitive yeast cells. Our working hypothesis is that, if the amino acid residue(s) involved in Pca1's cadmium specificity are substituted by those of copper-transporting P1B-type ATPase, these Pca1 alleles would export copper to confer copper resistance. These alleles could be selected by functional assays. This approach resulted in identification of several residues involved in cadmium selectivity of Pca1. Another research objective that was pursued was to gain mechanistic insights into cadmium sensing by a cadmium-responsive degradation signal that was identified within the Pca1 N-terminus. Site-directed mutagenesis and selection of randomly mutated signal domain in the context of a fusion to a drug efflux pump has mapped virtually all residues composing the degradation signal. These outputs were disseminated at two research conferences, FASEB Summer Research Conference on Transport ATPases and Redox Biology Center Annual Mini Symposium. PARTICIPANTS: This project has provided an opportunity for training and career development of four individuals, including one graduate student (100% effort), one postdoctoral fellow (100% effort), one research assistant professor (50% effort), and one undergraduate. TARGET AUDIENCES: The research outcomes would provide new information to scientists in the fields of metal detoxification, ATPase, and protein expression control. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our research progress provides several layers of novel insights into the mechanisms of action and regulation of P1B-type ATPases. Given that cadmium is a widespread environmental contaminant and known to be implicated in a number of human disorders, including kidney disease, cancer, and endocrine disruption, our study also underpins potential biotechnological applications for limiting human consumption of cadmium. We expect that characterization of cadmium-selective exporters ultimately facilitate development of methods for the prevention of cadmium exposure, treatment of cadmium-related diseases, and efficient remediation of cadmium from the environment. Collectively, the outcomes of this project would have broad and significant impacts on biology, food safety and human heath.
Publications
- No publications reported this period
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