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Mining Mitochondrial DNA for New Genes

05/18/2016
Amber Dance, PhD

It turns out that mitochondrial DNA contains many more genes than scientists initially thought, with tiny peptide-encoding ORFs nestled within bigger genes. This finding launched a new field of genetics. Learn more...


 The location of humanin within the mitochondrial 16S rRNA code.

Image courtesy of Pinchas Cohen.

Pinchas Cohen.

SHLPS within the mitochondrial genome (2).

For decades, scientists thought they had a handle on the mitochondrial chromosome: 13 genes for proteins, two for rRNAs and 22 for tRNAs, all tightly packed with no introns. “That’s a lot to ask of one small DNA,” said Pinchas “Hassy” Cohen, a mitochondrial biologist at the University of Southern California (USC). “And yet, on top of that, it appears to contain small open reading frames that are hidden inside the other genes.”

Cohen has discovered dozens of genes for short peptides nestled within the known mitochondrial genes like tiny Russian dolls. In two recent papers, Cohen and colleagues described seven such genes, encoding peptides 16-38 amino acids long, with roles regulating metabolism and either promoting or quelling apoptosis (1,2).

Cohen suspects that mitochondria use these small peptides to communicate their status to the cell and body. Several seem to be relevant to aging--a time when mitochondria begin to struggle--and aging-associated diseases. Already, the company he co-founded, CohBar, is developing one peptide, MOTS-c, as a potential treatment for obesity, type II diabetes, and fatty liver disease.

Oh, The Humanity

The story of mitochondrial derived peptides (MDPs) starts more than a decade ago when Japanese researchers were hunting for genes that protected cells from amyloid-beta, a toxic molecule found in the brains of people with Alzheimer’s disease. They found one and named it humanin, in the hopes it would restore “humanity” to people with dementia. Surprisingly, it resided within the gene for mitochondrial 16S rRNA (3,4). Simultaneously, Cohen’s lab and another group also cloned humanin (5,6).

The idea of additional tiny genes in the mitochondrial chromosome initially met with surprise and disbelief, but humanin’s location was eventually accepted. Cohen dropped his lab’s previous interests to devote the group entirely to finding other MDPs. Using bioinformatics tools to scan the human mitochondrial genome, they identified numerous possible ORFs. The precise number depends on how one sets the parameters, Cohen said—for example, whether he allows alternative start codons and the length permitted for an ORF. Based on his analyses, there could be up to hundreds of MDP genes, opening up a whole new field of genetics. In some cases, they’re translated in the mitochondria; other times they use the cell’s main protein production systems.

“The discovery that there are more genes in the mitochondrial genome was unexpected,” said David Sinclair, a geneticist at Harvard Medical School and co-founder of CohBar. “It could be just the tip of the iceberg, with potentially more than 50 other peptides coming from mitochondria.” Sinclair has found thousands of similar peptide genes in the nuclear genome.

Skinny Mice

In the mitochondrion’s 12S rRNA gene, Cohen and Changhan (David) Lee at USC discovered the gene for a 16-amino acid peptide they christened MOTS-c, for mitochondrial ORF of the 12S rRNA, as the group reported in Cell Metabolism in March, 2015 (1). (MOTS-a and -b are still under investigation.)

Once they find these small ORFs, Cohen and his team poke around somewhat blindly to discover their functions. One clue in this case, was that the mitochondrial 12S rRNA linked to diabetes (7,8). By treating HEK293 cells with MOTS-c and analyzing gene expression and the metabolome, Lee determined that it affected genes involved in cellular metabolism and inflammation, as well as numerous metabolites. In particular, it stimulated glycolysis.

To investigate the whole-body actions of MOTS-c, the researchers injected it into mice. The animals lost a bit of weight despite eating no differently. Compared to control mice, they had lower blood glucose levels, and elevated insulin sensitivity. Mice (and people) tend to become more insulin-resistant as they age, but one week of MOTS-c treatment restored the muscles of year-old mice to the insulin sensitivity seen in three-month-olds. When the authors treated mice on a high-fat diet, MOTS-c prevented obesity.

According to Cohen, the interesting thing about MOTS-c is that it contains the site of a single nucleotide polymorphism associated with longevity in Japanese people (9). “We are excited about the possibility that it’s a pro-longevity hormone,” he said.

Though Cohen does not think regular MOTS-c injections are the right solution for people out to lose a few pounds, he and his colleagues at CohBar believe it could be beneficial for people with more serious metabolic conditions like diabetes.

Schlepping Through the Mitochondrial Genome

By trawling the 16S rRNA gene, Lee, Cohen, and colleagues found six new MDPs, which they reported in the April 2016 issue of Aging (2). In homage to the 16S MDP humanin, the scientists christened the group small humanin-like peptides, or SHLPs, pronounced “schlepps.” “It’s an opportunity to have some fun with the Yiddish words,” quipped Cohen, who is Jewish.

The researchers managed to generate antibodies against five of the six SHLPs and found that all were expressed in mouse tissues. To test if they were protective like their namesake, the researchers incubated each peptide with starving cell cultures. SHLP2 and SHLP3 both delayed apoptosis, even in cultures exposed to toxins such as the caspase activator staurosporine, so the authors examined them more carefully. SHLP6, in contrast, promoted cellular suicide in cancerous cells.

In cultured cells, SHLP2 and SHLP3 sped up mitochondrial oxygen use and ATP production, and prevented production of dangerous reactive oxygen species. In mice, SHLP2 but not SHLP3 enhanced insulin sensitivity. It also protected neurons against amyloid-beta, like humanin.

Notably, deletions or mutations in the mitochondrial 16S rRNA are often associated with age-related macular degeneration, said Cohen (10), pointing to a possible role for SHLPs in aging.

CohBar expects to move a MOTS-c-based treatment into clinical trials within two years, and has licensed the SHLPs, too. SHLP6, for example, might treat cancer, and SHLP2 might lead to an Alzheimer’s therapy. CEO Simon Allen hopes MDPs could be beneficial for a variety of diseases of aging, but he has even bigger dreams. Levels of humanin, MOTS-c, and SHLP2 all drop as animals age—so might the peptides extend lifespan in general? “That’s a much longer trial, as you could imagine,” said Allen.

References

(1) Lee C et al. The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metab. 2015 Mar 3;21(3):443-54.

(2) Cobb LJ et al. Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers. Aging. 2016 Apr;8(4). Epub 2016 Apr 10.

(3) Hashimoto Y et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Abeta. Proc Natl Acad Sci U S A. 2001 May 22;98(11):6336-41.

(4) Hashimoto Y et al. Mechanisms of neuroprotection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein. Biochem Biophys Res Commun. 2001 May 4;283(2):460-8.

(5) Guo B et al. Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature. 2003 May 22;423(6938):456-61.

(6) Ikonen M et al. Interaction between the Alzheimer’s survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):13042-7. http://www.pnas.org/content/100/22/13042.long

(7) Yu Y et al. [Mutations of mitochondrial 12S rRNA gene in type 2 diabetes]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2001 Oct;18(5):388-90. http://www.ncbi.nlm.nih.gov/pubmed/11592049

(8) Mezghani N et al. A maternally inherited diabetes and deafness patient with the 1S rRNS m.1555A>G and the ND1 m.3308T>C mutations associated with multiple mitochondrial deletions. Biochem Biophys Res Commun. 2013 Feb 22;431(4):670-4.

(9) Fuku N et al. The mitochondrial-derived peptide MOTS-c: a player in exceptional longevity? Aging Cell. 2015 Dec;14(6):921-3.

(10) Ferrington DA et al. Increased retinal mtDNA damage in the CFH variant associated with age-related macular degeneration. Exp Eye Res. 2016 Apr;145:269-77.