Model of cytochrome c oxidase biogenesis

COX is the terminal oxidase of the respiratory chain. COX deficiency is the major cause of mitochondrial encephalomyopathies in humans. 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.

Translational regulation of cytochrome c oxidase biogenesis in S. cerevisiae

Translational regulation of COX assembly. COX is a hetero-oligomeric enzyme formed by subunits encoded in the nuclear and the mitochondrial DNA. Because COX contain highly reactive heme A and copper prosthetic groups, the biogenesis of this enzyme must be tightly regulated to prevent the accumulation of pro-oxidant assembly intermediates. Over the last few years we have used the yeast Saccharomyces cerevisiae to discover the existence of a negative feedback translational regulatory system. This system coordinates the synthesis of Cox1, a mtDNA-encoded catalytic subunit containing heme A and copper centers, with its assembly into the holoenzyme. With support from NIH and MDA, we identified a COX1 mRNA-specific translation activator, Mss51, as the key element of the system. Mss51 is a bi-functional protein that interacts with the 5’UTR of COX1 mRNA to promote translation and subsequently interacts with the newly synthesized Cox1 protein to facilitate its stability in pre-assembly complexes. Mss51 does not act alone. The general mitochondrial Hsp70 chaperone Ssc1 and the COX specific chaperones Cox14 and Cox25 dynamically interact with Mss51-containing complexes to coordinate Cox1 synthesis and assembly, and to facilitate Mss51 recycling between its two functions. More recently, we discovered that Mss51 binds heme. This finding, recently published in Cell Metabolism, has provided a key element for a regulatory mechanism that coordinates assembly of COX, the major oxygen-consuming mitochondrial enzyme, with heme and oxygen availability for respiration and aerobic energy production. Some ongoing projects include include: (i) Identification and characterization of the heme A insertase/s; (ii) Regulation of Mss51 function by Ssc1 (iii) Regulation of COX biogenesis by oxygen and reactive oxygen species.

Role of tween CX₉C-motif proteins in redox balance and copper delivery to COX. Although several mitochondrial COX copper chaperones are already known, there are essential gaps in our knowledge regarding how copper reaches mitochondria, how copper is transported from a matrix pool to the intermembrane space, how copper is distributed to COX and mt-Sod1 (Cu-Zn superoxide dismutase) and how the metallation state of these two enzymes regulates cellular copper homeostasis. We have discovered two novel conserved mitochondrial copper chaperones, Cmc1 and Cmc2, from the twin-CX₉C-motif family, required for the metallation of COX and also for the copper metallation of the mitochondrial intermembrane space portion of Sod1. We are currently working toward understanding how copper is partitioned between the two enzymes. Additionally, we continue the functional characterization of key CX₉C-motif proteins in COX assembly in yeast and human cell lines.

Role of COX assembly factors in human cells. Most COX biogenetic factors were initially discovered in yeast but they have human homologues whose functions are in many instances poorly understood. We are taking advantage of the new advances in gene editing technology to create human cell lines knockout for specific COX assembly factors. The KO cell lines are subsequently characterized and used to express mutant or tagged versions of KO genes. Using this approach, we have already published in Human Molecular Genetics the characterization of human COX20 as a COX2 chaperone that assist the copper metallation of this subunit by the SCO1/2 proteins.

Mitochondrial physiology and biogenesis in cellular stress and aging

Metabolic and mitochondrial abnormalities are a prominent feature of aging and neurodegeneration. However, the literature reports conflicting results concerning the extent and causality of the aging associated aerobic energy production decline and mitochondrial ROS-induced damage, as well as their interplay with nutritional cues. Single cell models have provided key information concerning mechanisms of aging and neurodegeneration. In collaboration with Dr. G. Shadel (Yale University, USA)), we have gained insight into the mechanism by which tor1 mutations extend yeast lifespan and have characterized the role of mitochondrial respiration in regulating yeast lifespan and its extension by caloric restriction (reported in two Cell Metabolism papers). We are now testing the hypothesis that mitochondrial respiration and ROS signaling interplay with nutritional cues and perform complementary roles on regulating the manifestation of age-induced cellular proteotoxicities.

We have developed novel transgenic yeast expressing human neurodegenerative disease-relevant proteins that recapitulate key features of neuronal proteotoxicity and have facilitated the elucidation of basic cellular mechanisms of toxicity triggered by human neurotoxic proteins. For example, we have shown that proteotoxicity can be suppressed by enhancement of mitochondrial biogenesis or by overexpressing the enzymes in the NAD⁺ salvage biosynthetic pathway. In collaboration with Dr. Grace Zhai (Molecular & Cellular Pharmacology, University of Miami), we have observed that similar effects are observed in Drosophila models of polyglutamine disorders. Results from ongoing experiments indicate that under stress, these proteins act as molecular chaperones to combat proteotoxicity.

Role of the DEAD-box helicase Mrh4 on the biogenesis of the S. cerevisiae mitochondrial ribosome

Over the last few years we have become very interested in the biogenesis of the mitochondrial ribosomes. The process is complicated by the fact that the two mitoribosomal RNAs (rRNAs) are universally mitochondrion-encoded whereas all ribosomal proteins (with a single exception in yeast) are encoded in the nuclear DNA. Its biomedical importance is highlighted by the fact that mutations affecting genes encoding mitochondrial ribosomal subunits are responsible for infantile multisystemic mitochondrial diseases, frequently involving encephalomyopathy and hypertrophic cardiomyopathy. Despite their biological and biomedical relevance, the knowledge on the molecular details of the assembly pathway and the factors involved in the biogenesis of mitoribosomes is still very limited. We have started searching for mitoribosome assembly factors and identified the first two DEAD-box helicases (Mrh4 in yeast and DDX28 human), involved in the process. The work on Mrh4 was recently published in Cell Metabolism and the work on DDX28 is been revised for Cell Reports. Both proteins are essential during late stages of assembly of the large mitoribosomal subunit. In addition, human DDX28 is part of RNA granules, a compartment localized near the mitochondrial nucleoids, where we propose mitoribosome assembly largely occurs. Our ambitious goal is to precisely characterize the steps and factors involved in the yeast and human mitoribosome assembly pathway.

Dynamic Rearrangements of MRC Complexes

The mitochondrial respiratory chain (MRC) assembly and function involve the organization of its constitutive complexes in supercomplexes or respirasomes. It is believed that supercomplexes have an important functional role in cellular bioenergetics by optimizing electron transfer, proton pumping and controlling the formation of reactive intermediates. We are investigating the players and mechanisms involved in MRC supercomplex assembly using yeast and cultured human cells as research models. In collaboration with Dr. Cristina Ugalde (Spain), we have recently reported the first human mitochondrial respirasome assembly pathway, which involves a paradigm-shifting model. Our data indicate that respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. These studies, published in two Cell Metabolism papers, are shedding light onto the structural interdependences between respiratory chain complexes.

Current projects aim to refining the proposed assembly pathway in human cells derived from controls and patients with mutations in respiratory chain subunits and assembly factors, characterizing the role several respiratory chain assembly factors play on respirasome biogenesis and investigating how the pathway is regulated by oxygen tension and oxidative stress.