APOPT1/COA8 assists COX assembly and is oppositely regulated by UPS and ROS.

Loss-of-function mutations in APOPT1, a gene exclusively found in higher eukaryotes, cause a characteristic type of cavitating leukoencephalopathy associated with mitochondrial cytochrome c oxidase (COX) deficiency. Although the genetic association of APOPT1 pathogenic variants with isolated COX defects is now clear, the biochemical link between APOPT1 function and COX has remained elusive. We investigated the molecular role of APOPT1 using different approaches. First, we generated an Apopt1 knockout mouse model which shows impaired motor skills, e.g., decreased motor coordination and endurance, associated with reduced COX activity and levels in multiple tissues. In addition, by achieving stable expression of wild-type APOPT1 in control and patient-derived cultured cells we ruled out a role of this protein in apoptosis and established instead that this protein is necessary for proper COX assembly and function. On the other hand, APOPT1 steady-state levels were shown to be controlled by the ubiquitination-proteasome system (UPS). Conversely, in conditions of increased oxidative stress, APOPT1 is stabilized, increasing its mature intramitochondrial form and thereby protecting COX from oxidatively induced degradation.

Assembly of mammalian oxidative phosphorylation complexes I-V and supercomplexes.

The assembly of the five oxidative phosphorylation system (OXPHOS) complexes in the inner mitochondrial membrane is an intricate process. The human enzymes comprise core proteins, performing the catalytic activities, and a large number of 'supernumerary' subunits that play essential roles in assembly, regulation and stability. The correct addition of prosthetic groups as well as chaperoning and incorporation of the structural components require a large number of factors, many of which have been found mutated in cases of mitochondrial disease. Nowadays, the mechanisms of assembly for each of the individual complexes are almost completely understood and the knowledge about the assembly factors involved is constantly increasing. On the other hand, it is now well established that complexes I, III and IV interact with each other, forming the so-called respiratory supercomplexes or 'respirasomes', although the pathways that lead to their formation are still not completely clear. This review is a summary of our current knowledge concerning the assembly of complexes I-V and of the supercomplexes.

MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase.

The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: Characterization of aggregates and factors affecting aggregation.

We investigated the influence of pH and sodium chloride concentration on aggregation kinetics of a monoclonal antibody. Aggregation was induced by sodium chloride addition at low pH. Protein conformation before and after salt addition was determined as well as the reversibility of aggregation. Aggregation was monitored at pH values between 2 and 7 with NaCl up to 1.5M by turbidity measurement and size-exclusion chromatography. Particle size distribution was assessed by using size-exclusion chromatography as well as nanoparticle tracking analysis and flow imaging microscopy. Structural changes were monitored by circular dichroism, Fourier transform infrared and fluorescence spectroscopy. Thermal stability was measured by differential scanning fluorimetry. Aggregation propensity was maximal at low pH and high ionic strength. While thermal stability decreased with pH, the secondary structure remained unchanged down to pH 3.5 and up to 1.5M NaCl. Precipitated protein could be largely reverted to monomers by dilution into salt-free buffer. The re-solubilized antibody was indistinguishable in structure, solubility and monodispersity from the unstressed protein. Also, binding to Protein A was steady. Aggregation could be reduced in the presence of trehalose. The results suggest a reversible aggregation mechanism characterized by a limited change in tertiary structure at low pH and a subsequent loss of colloidal stability resulting from electrostatic repulsion once salt is added to the sample. The experimental setup is robust and allows high-throughput quantification of the effect of additives on aggregation kinetics.