Biology subjects

Cobine, P. A.

Publications and source records attributed to Cobine, P. A..

6 recordsLinked to original sources

bioRxiv2026

Mitochondrial copper stabilizes lipoylated TCA cycle proteins to sustain metabolism and proliferation

Copper (Cu) is an essential cofactor for mitochondrial cytochrome c oxidase, yet whether it directly regulates mitochondrial metabolism beyond respiration remains unclear. Here we show that mitochondrial Cu, delivered by SLC25A3, is required to maintain the stability of lipoylated TCA cycle proteins. Loss of Slc25a3 or pharmacological Cu depletion selectively destabilized the lipoylated E2 subunits of mitochondrial dehydrogenases and the lipoylation enzymes LIPT1 and LIPT2, an effect not reproduced by acute electron transport chain inhibition. Mechanistically, we find that Cu directly engages the reduced lipoyl moiety using chemical probes and synthetic peptide approaches. Cu depletion impaired PDH and OGDH activity, rewired TCA cycle metabolism, and imposed a dependence on pyruvate carboxylase for anaplerosis. This metabolic defect depleted aspartate, suppressed mTORC1 signaling, and limited proliferation. Conversely, selective delivery of Cu to the mitochondria restored lipoylation, TCA cycle function, and cell growth. Together, these findings identify mitochondrial Cu as a structural regulator of the lipoylation machinery and reveal a direct link between Cu homeostasis and central carbon metabolism.

cell biology↗

bioRxiv2024

Copper trafficking in Trypanosoma cruzi: the transcriptional response of candidates to balance toxicity and recruitment

Trypanosoma cruzi (Chagas disease) depends on acquiring nutrients and cofactors, like copper (Cu), from its hosts. Cu is essential for aerobic organisms, but it can also be toxic, so its transport and storage must be regulated. In the present study, we characterized the effects of changes in Cu availability on growth, intracellular ion content, and oxygen consumption. Our results show that Cu is essential for epimastigote proliferation and for metacyclogenesis, while intracellular amastigotes suffered from Cu stress during infection. We identify several genes potentially involved in Cu metabolism among which orthologs of the conserved P-type Cu ATPases involved in Cu export and loading of secreted enzymes were found and named TcCuATPase. TcCuATPase transcription is regulated during infective stages and by Cu availability in epimastigotes. No homologs were identified for the high affinity importer CTR1 instead we propose that the iron transport TcIT a ZIP family transporter is involved in Cu uptake based on its transcriptional response to Cu. Further canonical Cu targets (based on homology to yeast and mammals) such as the iron reductase TcFR and the cupro-oxidase TcFet3 are up regulated during infective stages and under intracellular Cu stress. We also demonstrated that Cu, iron, and heme metabolisms are related. In sum, Cu metabolism is essential in T. cruzi life cycle. Even though cytosolic Cu-chaperons are still missing, we propose a model for Cu transport and intracellular distribution in T. cruzi including conserved factors such as TcCuATPase and others such as TcFR and TcIT playing novel functions.

microbiology↗

bioRxiv2022

Complete functional analysis of type IV pilus components of a reemergent plant pathogen reveals neofunctionalization of paralog genes

Type IV pilus (TFP) is a multifunctional bacterial structure involved in twitching motility, adhesion, biofilm formation, as well as natural competence. Here, by mutagenesis and functional analysis, we dissected the roles of all genes required for TFP biosynthesis and regulation in the reemergent plant pathogenic fastidious prokaryote Xylella fastidiosa. This xylem-limited, insect-transmitted pathogen lives constantly under flow conditions and therefore is highly dependent on TFP for host colonization. In addition, TFP-mediated natural transformation is a process that impacts genomic diversity and environmental fitness. Ten out of the thirty-eight genes analyzed were essential for movement and natural competence. Interestingly, seven sets of paralogs exist, and mutations showed opposing phenotypes, indicating evolutionary neofunctionalization of subunits within TFP. The minor pilin FimT3 was the only protein exclusively required for natural competence. We determined that FimT3 (but not the other two FimT paralogs) is a DNA receptor that is conserved among X. fastidiosa strains and binds DNA non-specifically via an electropositive surface. Among plant pathogens, this gene was also found in the genome of strains of the plant associated Xanthomonadaceae family. Overall, we highlight here the complex regulation of TFP in X. fastidiosa, providing a blueprint to understand TFP in other bacteria living under flow conditions.

microbiology↗

bioRxiv2021

Mitochondrial dysfunction triggers secretion of the immunosuppressive factor α-fetoprotein

Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identify a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that results in atrophy of the spleen and thymus and causes a peripheral white blood cell deficiency. We demonstrate that the leukopenia is caused by -fetoprotein, which requires copper and the cell surface receptor CCR5 to promote white blood cell death. We further show that -fetoprotein expression is upregulated in several cell types upon inhibition of oxidative phosphorylation, including a muscle cell model of Barth syndrome. Collectively, our data argue that -fetoprotein secreted by bioenergetically stressed tissue suppresses the immune system, an effect which may explain the recurrent infections that are observed in a subset of mitochondrial diseases or in other disorders with mitochondrial involvement.

cell biology↗

bioRxiv2020

Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes

Mitochondrial carrier family (MCF/SLC25) proteins are selective transporters that maintain the mitochondrial metabolome. Here we combine computational, biochemical and phenotypic approaches to understand substrate selectivity of SLC25A3. In mammals, SLC25A3 transports both copper and phosphate, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2, which transports copper, and MIR1, which transports phosphate. To understand whether the ancestral state of this transporter was a single promiscuous transporter that duplicated and gained selectivity, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses reveal that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating that the gene duplication that created these paralogs occurred early in eukaryotic evolution. Frequent lineage-specific gene duplications and losses suggest that substrate specificity may be evolutionarily labile. To link this phylogenetic signal to protein function and resolve the residues involved in substrate selection, we used structural modelling and site-directed mutagenesis to identify PIC2 residues involved in copper and phosphate transport activities. Based on these analyses, we generated a Leu175Ala variant of mouse SLC25A3 that retains the ability to transport copper, but not phosphate, and rescues the cytochrome c oxidase defect in SLC25A3 knockout cells. Taken together, this work uses an evolutionary framework to uncover amino acids involved in substrate recognition by MCF proteins responsible for copper and phosphate transport.

evolutionary biology↗

bioRxiv2017

On the bioconversion of dietary carotenoids to astaxanthin and its distribution in the marine copepod, Tigriopus californicus

Red carotenoid-based coloration is widely distributed across marine and terrestrial animals and has taken a prominent role in studies of how phenotypic traits evolve in response to natural and sexual selection. Key to these studies is an understanding of the physiological mechanisms that give rise to red coloration, yet an ideal model system for such work has not been identified. The red marine copepod Tigriopus californicus is used as a model system for studies in ecotoxicology, genetics, and physiology, but the mechanisms involved in its bright red coloration have not been well studied. Like nearly all animals that display red carotenoid coloration, T. californicus likely convert yellow carotenoids present in their algal diet to red carotenoids. We conducted precursor/product feeding experiments to demonstrate that T. californicus bioconverts dietary carotenoids to the red carotenoid, astaxanthin. In separate treatment groups, copepods were fed carotenoids that are precursors to specific astaxanthin bioconversion pathways. We found that copepods from each precursor pigment group produced astaxanthin, and that the amount produced depended on which carotenoid was supplemented. We also describe the distribution of astaxanthin in developing egg sacs and show that the red color of the naupliar eyespot is not due to astaxanthin. We briefly discuss the potential of Tigriopus californicus to serve as a model system for the study of carotenoid metabolism in animals.

zoology↗