NEW YORK – With a large-scale screening and multiomics strategy, a Washington University- and University of Wisconsin at Madison-led team systematically characterized human cell lines missing mitochondrial proteins with yet-unknown functions, using the resulting resource to detect proteins behind three mitochondrial conditions.
"[H]undreds of mitochondrial proteins lack clear functions, and the underlying genetic basis for approximately 40 percent of mitochondrial disorders remains unresolved," co-senior and co-corresponding author David Pagliarini, a cell biology and physiology researcher affiliated with Washington University School of Medicine, the Morgridge Institute for Research, and the National Center for Quantitative Biology of Complex Systems, and his colleagues wrote in a report published Wednesday in Nature.
The researchers relied on CRISPR-Cas9-based editing — combined with liquid chromatography, tandem mass spectrometry, and gas chromatography-MS-based protein, lipid, and metabolite quantifications — to characterize 203 HAP1 human cell lines missing mitochondrial proteins encoded in the nuclear genome.
With levels for more than 8,400 proteins, almost 3,600 lipids, and 218 metabolites in the cell lines, combined with growth rate monitoring, the team tallied the effects of each mitochondrial protein in question, comparing the effects to mitochondrial proteins with known functions to come up with a "mitochondrial orphan protein multi-omics," or MITOMICS, tool.
"Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization," the authors reported, "and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases."
Along with analyses demonstrating that the dataset could be used to detect known mitochondrial protein functions, the investigators went on to use the MITOMICS tool to predict functions for other mitochondrial proteins with previously unknown functions, including those implicated in a handful of mitochondrial conditions.
"[T]his large dataset becomes one of a number in the field that collectively help us to devise better biomarkers and diagnostics for mitochondrial diseases," Pagliarini said in a statement.
In particular, the investigators found that cells missing a methyltransferase enzyme chaperone-coding "PIGY upstream open reading frame" (PYURF) contributes to complex I assembly and coenzyme Q biosynthesis, while the absence of PYURF is linked to a multisystemic condition caused by mitochondrial defects — results backed up with exome sequencing analyses on a child with metabolic acidosis, developmental delays, and alterations affecting muscle and brain structures that unearthed a PYURF frameshift variant.
Cells lacking another mitochondrial gene — the suspected zinc transporter-coding gene SLC30A9 — had features linked to lower-than-usual levels of mitochondrial ribosomes and oxidative phosphorylation pathway proteins.
The team also saw signs that alterations affecting a mitochondrial protein encoded by the RAB5IF gene can disrupt TMCO1. The latter gene has been implicated in an intellectual disability- and facial feature-related condition called cerebrofaciothoracic dysplasia, also known as craniofacial dysmorphism, skeletal anomalies, and mental retardation syndrome (CFSMR).
"Overall, these data inextricably link two poorly understood proteins, RAB5IF and TMCO1, thereby providing a new route to explore the function of each protein and understand the underlying pathophysiology of a debilitating disorder," the authors reported. "The partial localization of TMCO1 to the ER suggests that the connection between these proteins, and the etiology of CFSMR, may involve inter-organelle interaction."
The results point to the possibility of finding still other mitochondrial disease contributors by further delving into the MITOMICS data, Pagliarini explained, noting that "[e]very time we discover a function of a new protein, it gives us a new opportunity to target a pathway therapeutically."
"Our long-term goal is to understand mitochondria at sufficient depth to be able to intervene therapeutically, which we can't do yet," he said.