Putative Concussion Biomarkers Identified in Adolescent Male Athletes Using Targeted Plasma Proteomics

Sport concussions can be difficult to diagnose and if missed, they can expose athletes to greater injury risk and long-lasting neurological disabilities. Discovery of objective biomarkers to aid concussion diagnosis is critical to protecting athlete brain health. To this end, we performed targeted proteomics on plasma obtained from adolescent athletes suffering a sports concussion. A total of 11 concussed male athletes were enrolled at our academic Sport Medicine Concussion Clinic, as well as 24 sex-, age- and activity-matched healthy control subjects. Clinical evaluation was performed and blood was drawn within 72 h of injury. Proximity extension assays were performed for 1,472 plasma proteins; a total of six proteins were considered significantly different between cohorts (P < 0.01; five proteins decreased and one protein increased). Receiver operating characteristic curves on the six individual protein biomarkers identified had areas-under-the-curves (AUCs) for concussion diagnosis ≥0.78; antioxidant 1 copper chaperone (ATOX1; AUC 0.81, P = 0.003), secreted protein acidic and rich in cysteine (SPARC; AUC 0.81, P = 0.004), cluster of differentiation 34 (CD34; AUC 0.79, P = 0.006), polyglutamine binding protein 1 (PQBP1; AUC 0.78, P = 0.008), insulin-like growth factor-binding protein-like 1 (IGFBPL1; AUC 0.78, P = 0.008) and cytosolic 5'-nucleotidase 3A (NT5C3A; AUC 0.78, P = 0.009). Combining three of the protein biomarkers (ATOX1, SPARC and NT5C3A), produced an AUC of 0.98 for concussion diagnoses (P < 0.001; 95% CI: 0.95, 1.00). Despite a paucity of studies on these three identified proteins, the available evidence points to their roles in modulating tissue inflammation and regulating integrity of the cerebral microvasculature. Taken together, our exploratory data suggest that three or less novel proteins, which are amenable to a point-of-care immunoassay, may be future candidate biomarkers for screening adolescent sport concussion. Validation with protein assays is required in larger cohorts.

The mitochondrial copper chaperone COX11 has an additional role in cellular redox homeostasis

Mitochondria are sites of cellular respiration, which is accompanied by the generation of dangerous reactive oxygen species (ROS). Cells have multiple mechanisms to mitigate the dangers of ROS. Here we investigate the involvement of the COX complex assembly chaperone COX11 (cytochrome c oxidase 11) in cellular redox homeostasis, using homologs from the flowering plant Arabidopsis thaliana (AtCOX11) and yeast Saccharomyces cerevisiae (ScCOX11). We found that AtCOX11 is upregulated in Arabidopsis seedlings in response to various oxidative stresses, suggesting a defensive role. In line with this, the overexpression of either AtCOX11 or ScCOX11 reduced ROS levels in yeast cells exposed to the oxidative stressor paraquat. Under normal growth conditions, both Arabidopsis and yeast COX11 overexpressing cells had the same ROS levels as the corresponding WT. In contrast, the COX11 knock-down and knock-out in Arabidopsis and yeast, respectively, significantly reduced ROS levels. In yeast cells, the ScCOX11 appears to be functionally redundant with superoxide dismutase 1 (ScSOD1), a superoxide detoxifying enzyme. The ΔSccox11ΔScsod1 mutants had dramatically reduced growth on paraquat, compared with the WT or single mutants. This growth retardation does not seem to be linked to the status of the COX complex and cellular respiration. Overexpression of putatively soluble COX11 variants substantially improved the resistance of yeast cells to the ROS inducer menadione. This shows that COX11 proteins can provide antioxidative protection likely independently from their COX assembly function. The conserved Cys219 (in AtCOX11) and Cys208 (in ScCOX11) are important for this function. Altogether, these results suggest that COX11 homologs, in addition to participating in COX complex assembly, have a distinct and evolutionary conserved role in protecting cells during heightened oxidative stress.

Copper depletion modulates mitochondrial oxidative phosphorylation to impair triple negative breast cancer metastasis

Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials.

The Modulation of SCO2730/31 Copper Chaperone/Transporter Orthologue Expression Enhances Secondary Metabolism in Streptomycetes

Streptomycetes are important biotechnological bacteria that produce several clinically bioactive compounds. They have a complex development, including hyphae differentiation and sporulation. Cytosolic copper is a well-known modulator of differentiation and secondary metabolism. The interruption of the Streptomyces coelicolor SCO2730 (copper chaperone, SCO2730::Tn5062 mutant) blocks SCO2730 and reduces SCO2731 (P-type ATPase copper export) expressions, decreasing copper export and increasing cytosolic copper. This mutation triggers the expression of 13 secondary metabolite clusters, including cryptic pathways, during the whole developmental cycle, skipping the vegetative, non-productive stage. As a proof of concept, here, we tested whether the knockdown of the SCO2730/31 orthologue expression can enhance secondary metabolism in streptomycetes. We created a SCO2730/31 consensus antisense mRNA from the sequences of seven key streptomycetes, which helped to increase the cytosolic copper in S. coelicolor, albeit to a lower level than in the SCO2730::Tn5062 mutant. This antisense mRNA affected the production of at least six secondary metabolites (CDA, 2-methylisoborneol, undecylprodigiosin, tetrahydroxynaphtalene, α-actinorhodin, ε-actinorhodin) in the S. coelicolor, and five (phenanthroviridin, alkylresorcinol, chloramphenicol, pikromycin, jadomycin G) in the S. venezuelae; it also helped to alter the S. albus metabolome. The SCO2730/31 consensus antisense mRNA designed here constitutes a tool for the knockdown of SCO2730/31 expression and for the enhancement of Streptomyces’ secondary metabolism.

Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the cbb3-Type Cytochrome c Oxidase in the Enteric Pathogen Campylobacter jejuni

Bacterial C-type haem-copper oxidases in the cbb3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb3-oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential (cj1483c and cj1486c) or important (cj1484c and cj1485c) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis. Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI (cj1155c) and ccoS (cj1154c) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCuAC (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c–1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA (cj1161c), or copZ (cj1162c) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen.