The formation and regulation of macromolecular complexes provides the backbone of aerobic energy production by the oxidative phosphorylation system within the mitochondrial membranes. Our parent grant is focused on cytochrome c oxidase (COX), the last mitochondrial respiratory chain enzyme, whose biogenesis involves a sophisticated network of dynamic protein-protein interactions that we are just beginning to disclose. COX is a heme a-copper-containing multimeric enzyme formed by polypeptides of nuclear and mitochondrial genetic origins. In the parent grant, we have revealed an assembly-feedback regulatory mechanism by which the synthesis of the key mtDNA-encoded and heme a-containing Cox1 subunit is regulated by the availability of its assembly partners in Saccharomyces cerevisiae. In this way, Cox1 synthesis and assembly are tightly coordinated. The central element of this regulatory system is the bi-functional COX1 mRNA translational activator and Cox1 chaperone Mss51 which is conserved from yeast to humans. During Cox1 synthesis, Mss51 interacts with the elongating polypeptide in translational complexes and in post-translational pre-assembly Cox1- containing high molecular weight complexes. These complexes are stabilized by two conserved small, single transmembrane domain proteins, Cox14 and the newly identified Cox25. At a point concurrent with Cox1 hemylation and/or the incorporation of subunit Cox5, Mss51 is released and is available for further rounds of translation. Cox14 and Cox25 instead, remain bound to Cox1 and other assembly chaperones to promote late stages of COX assembly. Recent observations suggest a similar mechanism exists in human cells. The parent grant explores the role of the human MSS51 homologue in COX assembly. Now, homologues of Cox14 and Cox25 have been also identified. The central hypothesis of this revision is the existence in human cells of an assembly-dependent regulation of Cox1 synthesis, in which MSS51, COX14 and COX25-containing multichaperone complexes act to coordinate Cox1 synthesis and multistep COX assembly.
Two specific aims are proposed to characterize interacting partners of Cox14 and Cox25 and the conservation of these interactions from yeast to humans.
Aim # 1 - Characterize MSS51, COX14 and COX25 knockout human cell lines created by a using a TALENs (transcription activator-like effector nucleases) approach.
Aim # 2 - Characterize macromolecular multi-chaperone COX assembly complexes involving human MSS51, COX14 and COX25

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 human cultured cells to study the functions of MSS51, COX14 and COX25, three proteins that in yeast form dynamic macromolecular complexes with COX subunits, other COX specific assembly factors and general mitochondrial chaperones to coordinate COX biogenesis. The approach will integrate the creation and characterization of gene knockout human cell lines using an innovative TALENs (transcription activator-like effector nucleases) approach. Mutations in human COX14 are responsible for COX related mitochondrial disorders and human MSS51 and COX25 could potentially be disease genes. To gain knowledge on the function of these proteins and the network of interactions in which they operate 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 #
3R01GM071775-07S2
Application #
8448954
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (MI))
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
$122,633
Indirect Cost
$42,633
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
Kumar, Akhilesh; Birnbaum, Michael D; Patel, Devang M et al. (2016) Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response. Cell Death Dis 7:e2378
Bohovych, Iryna; Kastora, Stavroula; Christianson, Sara et al. (2016) Oma1 Links Mitochondrial Protein Quality Control and TOR Signaling To Modulate Physiological Plasticity and Cellular Stress Responses. Mol Cell Biol 36:2300-12
Soto, Iliana C; Barrientos, Antoni (2016) Mitochondrial Cytochrome c Oxidase Biogenesis Is Regulated by the Redox State of a Heme-Binding Translational Activator. Antioxid Redox Signal 24:281-98
Abrams, Alexander J; Hufnagel, Robert B; Rebelo, Adriana et al. (2015) Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder. Nat Genet 47:926-32
Tu, Ya-Ting; Barrientos, Antoni (2015) The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly. Cell Rep :
Ruetenik, Andrea; Barrientos, Antoni (2015) Dietary restriction, mitochondrial function and aging: from yeast to humans. Biochim Biophys Acta 1847:1434-47
Neal, Sonya E; Dabir, Deepa V; Tienson, Heather L et al. (2015) Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway. J Biol Chem 290:20804-14
De Silva, Dasmanthie; Tu, Ya-Ting; Amunts, Alexey et al. (2015) Mitochondrial ribosome assembly in health and disease. Cell Cycle 14:2226-50
Tigano, Marco; Ruotolo, Roberta; Dallabona, Cristina et al. (2015) Elongator-dependent modification of cytoplasmic tRNALysUUU is required for mitochondrial function under stress conditions. Nucleic Acids Res 43:8368-80
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

Showing the most recent 10 out of 35 publications