Fight Aging!

A paper published last month outlines recent progress on allotopic expression of mitochondrial genes carried out by the SENS Research Foundation team. Allotopic expression is the name given to the process of putting copies of mitochondrial genes into the nuclear genome, suitably altered to allow proteins to be generated and shipped back to the mitochondria where they are needed. Mitochondria replicate like bacteria, and some forms of stochastic mitochondrial DNA damage can make mitochondria both dysfunctional and able to outcompete their undamaged peers. This is thought to be an important contribution to aging, resulting a small but damaging population of cells that are overtaken by broken mitochondria and which export harmful reactive molecules into the surrounding tissues.

Having a backup supply of mitochondrial proteins can in principle block these consequences of mitochondrial DNA damage, and thus remove this contribution to the aging process. Proof of concept has been demonstrated for a few of the thirteen proteins needed, and work proceeds on the rest. As noted here, one of the challenges in this project is that mitochondrial genetic machinery is of a different evolutionary origin to that of the cell nucleus, and thus the efficient production of equivalent proteins from nuclear genes is a much more challenging process than would otherwise be the case.

While the vast majority of mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol, and imported into the mitochondrion, 13 core subunits of respiratory complexes are encoded by the reduced mitochondrial genome and synthesized within the mitochondrial matrix. Mutations in these 13 genes (or their associated non-protein-coding genes) tend to be especially severe, as all 13 proteins are core subunits of the oxidative phosphorylation chain, and any disruption to subunit structure, stability, or function may have grave biochemical and physiological consequences. Gene therapy to target affected mitochondrial subunits is a promising alternative strategy which circumvents some of the technical challenges faced by the above approaches. One issue that remains, however, relates to the prokaryotic origin of the organelle. Translation within the mitochondrion deviates from the universal genetic code, utilizing machinery and codon frequencies more similar to its α-proteobacterial ancestry than to the mammalian nuclear genome.

Subsequently, allotopic expression has been suggested as a therapeutic tool to genetically remedy deleterious mitochondrial DNA mutations through nuclear complementation of the affected genes. A critical, but often-overlooked consideration in these nuclear relocation studies is the influence of the primary coding sequence on protein production. The vast majority of these previous studies have utilized what may be considered "minimally-recoded" mitochondrial genes. While making these codon changes is essential to maintain amino acid sequence integrity during cytosolic translation, this minimal approach fails to account for other elements of primary sequence which can critically influence both gene and protein expression.

Many commercial algorithms have therefore been developed to determine the optimal sequence and conditions for expression of a gene from a particular host. Though there are concerns regarding the use of codon optimization to increase homologous expression of a nuclear gene, such as the generation of novel or immunogenic peptides or structural perturbations in the encoded protein, codon optimization continues to be widely utilized for the production of biotherapeutics. Applying this principle to allotopic expression, we hypothesize that, given the bacterial origin of the mitochondrial genome, the coding sequences of minimally-recoded mitochondrial genes are dissimilar from nuclear genes and are inefficiently translated by nuclear machinery, therefore resulting in poor allotopic expression.

Here we employed codon optimization as a tool to re-engineer the protein-coding genes of the human mitochondrial genome for robust, efficient expression from the nucleus. All 13 codon-optimized constructs exhibited substantially higher protein expression than minimally-recoded genes when expressed transiently, and steady-state mRNA levels for optimized gene constructs were 5-180 fold enriched over recoded versions in stably-selected wildtype cells. Eight of thirteen mitochondria-encoded oxidative phosphorylation proteins maintained protein expression following stable selection, with mitochondrial localization of expression products. We also assessed the utility of this strategy in rescuing mitochondrial disease cell models and found the rescue capacity of allotopic expression constructs to be gene specific. Allotopic expression of codon optimized ATP8 in disease models could restore protein levels and respiratory function, however, rescue of the pathogenic phenotype for another gene, ND1, was only partially successful. These results imply that though codon-optimization alone is not sufficient for functional allotopic expression of most mitochondrial genes, it is an essential consideration in their design.

Link: https://doi.org/10.1016/j.redox.2020.101429