OMA1 protease eliminates arrested protein import intermediates upon mitochondrial depolarization.

Most mitochondrial proteins originate from the cytosol and require transport into the organelle. Such precursor proteins must be unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. We developed the cell line-based translocase clogging model to discover molecular mechanisms that resolve failed import events in humans. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by VCP/p97 and the proteasome.

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.

Defining mitochondrial protein functions through deep multiomic profiling.

Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. 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 and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.

Multi-Omic Single-Shot Technology for Integrated Proteome and Lipidome Analysis.

Mass spectrometry (MS) serves as the centerpiece technology for proteome, lipidome, and metabolome analysis. To gain a better understanding of the multifaceted networks of myriad regulatory layers in complex organisms, integration of different multiomic layers is increasingly performed, including joint extraction methods of diverse biomolecular classes and comprehensive data analyses of different omics. Despite the versatility of MS systems, fractured methodology drives nearly all MS laboratories to specialize in analysis of a single ome at the exclusion of the others. Although liquid chromatography-mass spectrometry (LC-MS) analysis is similar for different biomolecular classes, the integration on the instrument level is lagging behind. The recent advancements in high flow proteomics enable us to take a first step towards integration of protein and lipid analysis. Here, we describe a technology to achieve broad and deep coverage of multiple molecular classes simultaneously through multi-omic single-shot technology (MOST), requiring only one column, one LC-MS instrument, and a simplified workflow. MOST achieved great robustness and reproducibility. Its application to a Saccharomyces cerevisiae study consisting of 20 conditions revealed 2842 protein groups and 325 lipids and potential molecular relationships.

Large-Scale Multi-omic Analysis of COVID-19 Severity.

We performed RNA-seq and high-resolution mass spectrometry on 128 blood samples from COVID-19-positive and COVID-19-negative patients with diverse disease severities and outcomes. Quantified transcripts, proteins, metabolites, and lipids were associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many of which were involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a machine learning approach for prediction of COVID-19 severity.

A large-scale genome-lipid association map guides lipid identification.

Despite the crucial roles of lipids in metabolism, we are still at the early stages of comprehensively annotating lipid species and their genetic basis. Mass spectrometry-based discovery lipidomics offers the potential to globally survey lipids and their relative abundances in various biological samples. To discover the genetics of lipid features obtained through high-resolution liquid chromatography-tandem mass spectrometry, we analysed liver and plasma from 384 diversity outbred mice, and quantified 3,283 molecular features. These features were mapped to 5,622 lipid quantitative trait loci and compiled into a public web resource termed LipidGenie. The data are cross-referenced to the human genome and offer a bridge between genetic associations in humans and mice. Harnessing this resource, we used genome-lipid association data as an additional aid to identify a number of lipids, for example gangliosides through their association with B4galnt1, and found evidence for a group of sex-specific phosphatidylcholines through their shared locus. Finally, LipidGenie's ability to query either mass or gene-centric terms suggests acyl-chain-specific functions for proteins of the ABHD family.

Large-scale Multi-omic Analysis of COVID-19 Severity.

We performed RNA-Seq and high-resolution mass spectrometry on 128 blood samples from COVID-19 positive and negative patients with diverse disease severities. Over 17,000 transcripts, proteins, metabolites, and lipids were quantified and associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a comparative analysis with published data and a machine learning approach for prediction of COVID-19 severity.

Altered microglia and neurovasculature in the Alzheimer's disease cerebellum.

Traditionally regarded to coordinate movement, the cerebellum also exerts non-motor functions including the regulation of cognitive and behavioral processing, suggesting a potential role in neurodegenerative conditions affecting cognition, such as Alzheimer's disease (AD). This study aims to investigate neuropathology and AD-related molecular changes within the neocerebellum using post-mortem human brain tissue microarrays (TMAs). Immunohistochemistry was conducted on neocerebellar paraffin-embedded TMAs from 24 AD and 24 matched control cases, and free-floating neocerebellar sections from 6 AD and 6 controls. Immunoreactivity was compared between control and AD groups for neuropathological hallmarks (amyloid-ß, tau, ubiquitin), Purkinje cells (calbindin), microglia (IBA1, HLA-DR), astrocytes (GFAP) basement-membrane associated molecules (fibronectin, collagen IV), endothelial cells (CD31/PECAM-1) and mural cells (PDGFRß, αSMA). Amyloid-ß expression (total immunolabel intensity) and load (area of immunolabel) was increased by >4-fold within the AD cerebellum. Purkinje cell counts, ubiquitin and tau immunoreactivity were unchanged in AD. IBA1 expression and load was increased by 91% and 69%, respectively, in AD, with no change in IBA1-positive cell number. IBA1-positive cell process length and branching was reduced by 22% and 41%, respectively, in AD. HLA-DR and GFAP immunoreactivity was unchanged in AD. HLA-DR-positive cell process length and branching was reduced by 33% and 49%, respectively, in AD. Fibronectin expression was increased by 27% in AD. Collagen IV, PDGFRß and αSMA immunoreactivity was unchanged in AD. The number of CD31-positive vessels was increased by 98% in AD, suggesting the increase in CD31 expression and load in AD is due to greater vessel number. The PDGFRß/CD31 load ratio was reduced by 59% in AD. These findings provide evidence of molecular changes affecting microglia and the neurovasculature within the AD neocerebellum. These changes, occurring without overt neuropathology, support the hypothesis of microglial and neurovascular dysfunction as drivers of AD, which has implications on the neocerebellar contribution to AD symptomatology and pathophysiology.

Genetic determinants of gut microbiota composition and bile acid profiles in mice.

The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition. To address this, we used the Diversity Outbred (DO) stock, a population of genetically distinct mice derived from eight founder strains. We characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 DO mice maintained on a high-fat high-sucrose diet for ~22 weeks. Using quantitative trait locus (QTL) analysis, we identified several genomic regions associated with variations in both bacterial and BA profiles. Notably, we found overlapping QTL for Turicibacter sp. and plasma cholic acid, which mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs. This work illustrates how systems genetics can be utilized to generate testable hypotheses and provide insight into host-microbe interactions.

An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Strontium and bisphosphonate coated iron foam scaffolds for osteoporotic fracture defect healing.

The purpose of this work was to investigate new bone formation in macroporous iron foams coated with strontium (FeSr) or bisphosphonate (FeBiP) compared to plain iron foam (Fe) and empty defect in a critical size metaphyseal bone defect model in ovariectomized rats. 60 female rats were subjected to bilateral ovariectomy and multi-deficient diet for 3 months. A 4 mm wedge shaped metaphyseal osteotomy was created, fixed with a mini-plate and subsequently filled with Fe, FeSr, FeBiP or left empty. After 6 weeks, µCt analysis revealed a statistically significant increased bone formation at the implant interface in FeSr compared to FeBiP (p = 0.035) and Fe (p = 0.002), respectively. Increased mineralized tissue was also seen within the pores in FeSr (p = 0.023) compared to Fe. Histomorphometry revealed significantly increased bone formation at the implant interface in FeSr (p < 0.001) and FeBiP (p = 0.006) compared to plain Fe with increased osteoblast and decreased osteoclast activity in combination with increased BMP2 and decreased RANKL/OPG in immunohistochemistry. ToF-SIMS analysis showed overlapping Ca signals with Fe for both FeSr and FeBiP thereby indicating tissue in-growth into the scaffolds. In conclusion, iron foam with strontium or bisphosphonate coating are of further interest in metaphyseal fracture defects in osteopenic bone.