Cytochrome c oxidase (COX) deficiency is the most frequent cause of mitochondrial encephalomyopathies in humans and has also been associated to neurodegeneration and aging. A better understanding of COX biogenesis is essential for elucidating the molecular basis underlying these groups of diseases. The main objective of the proposed research is to investigate the players and mechanisms involved in COX biogenesis using the yeast Saccharomyces cerevisiae and cultured human cells as research models. Our long-term goal is to attain a complete understanding of the pathways leading to COX assembly and their components as a prerequisite to the development of therapies for the management of disorders associated with COX deficiencies. In the previous term of our grant we made significant progress in the understanding of how COX biogenesis is regulated. Eukaryotic COX is a multimeric enzyme formed by polypeptides of dual genetic origin (nuclear and mitochondrial) which assembly involves a large number of nuclear-encoded auxiliary factors. COX display a concerted accumulation of its constitutive subunits. Unassembled subunits bear a high risk to produce reactive oxygen species and their accumulation is limited by posttranslational degradation. We have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis. Our data support the existence of a regulatory mechanism by which the synthesis of mtDNA-encoded COX subunit 1 (Cox1) is regulated by the availability of its assembly partners in the yeast Saccharomyces cerevisiae. Thus, the central hypothesis of this proposal is the existence of a COX assembly dependent regulation of Cox1 synthesis, which unique properties, in turn, offer a means to catalyze multiple- subunit assembly. In S. cerevisiae, the regulatory system involves specific COX1 mRNA translational activators, specific Cox1 chaperones and assembly factors as well as general chaperones acting together in a sophisticated interplay that we are just beginning to characterize. The central element of the regulatory system is the bi-functional COX1 mRNA translational activator and Cox1 chaperone Mss51 which is conserved from yeast to humans.
Three specific aims are proposed to characterize recently identified new Mss51-interacting players (i.e. Ssc1 and Cox25) and levels of complexity (i.e. Mss51 is a heme binding protein) concerning the translational regulatory system in S. cerevisiae and its conservation from yeast to humans.
Aim # 1 - Disclose the mechanism and role of heme binding by Mss51 Aim #2 - Investigate the mechanism/s by which the interactions of Mss51 with mitochondrial chaperones regulate Cox1 synthesis Aim # 3 - Determine the functional equivalence of yeast and human Mss51

Public Health Relevance

Cytochrome c oxidase (COX) deficiency is the most frequent cause of mitochondrial encephalomyopathies in humans and has also been associated to neurodegeneration and aging. We will use the yeast Saccharomyces cerevisiae and human cultured cells to study the function of Mss51, a protein that plays dual functions in the synthesis of COX subunit 1 and in the assembly of this subunit into the COX holoenzyme. To gain knowledge on the function of this protein and the pathways in which it operates to regulate COX biogenesis is of great importance from a biological point of view and is expected to have an impact on our understanding of the pathogenic mechanisms underlying the above-mentioned kind of disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM071775-07S1
Application #
8589242
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Anderson, Vernon
Project Start
2006-02-01
Project End
2015-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
7
Fiscal Year
2013
Total Cost
$78,799
Indirect Cost
$27,403
Name
University of Miami School of Medicine
Department
Neurology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Hess, Kenneth C; Liu, Jingjing; Manfredi, Giovanni et al. (2014) A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity. FASEB J 28:4369-80
Bourens, Myriam; Boulet, Aren; Leary, Scot C et al. (2014) Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase. Hum Mol Genet 23:2901-13
De Silva, Dasmanthie; Fontanesi, Flavia; Barrientos, Antoni (2013) The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit. Cell Metab 18:712-25
Kursu, V A Samuli; Pietikainen, Laura P; Fontanesi, Flavia et al. (2013) Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae. Mol Microbiol 90:824-40
Barrientos, Antoni; Ugalde, Cristina (2013) I function, therefore I am: overcoming skepticism about mitochondrial supercomplexes. Cell Metab 18:147-9
Ocampo, Alejandro; Liu, Jingjing; Barrientos, Antoni (2013) NAD+ salvage pathway proteins suppress proteotoxicity in yeast models of neurodegeneration by promoting the clearance of misfolded/oligomerized proteins. Hum Mol Genet 22:1699-708
Diaz, Francisca; Barrientos, Antoni; Fontanesi, Flavia (2009) Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using blue native gel electrophoresis. Curr Protoc Hum Genet Chapter 19:Unit19.4
Barrientos, Antoni; Fontanesi, Flavia; Diaz, Francisca (2009) Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays. Curr Protoc Hum Genet Chapter 19:Unit19.3
Fontanesi, Flavia; Diaz, Francisca; Barrientos, Antoni (2009) Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using yeast models of OXPHOS deficiencies. Curr Protoc Hum Genet Chapter 19:Unit19.5
Barrientos, Antoni; Gouget, Karine; Horn, Darryl et al. (2009) Suppression mechanisms of COX assembly defects in yeast and human: insights into the COX assembly process. Biochim Biophys Acta 1793:97-107

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