Biology subjects

Jacobs, H. T.

Publications and source records attributed to Jacobs, H. T..

7 recordsLinked to original sources

bioRxiv2023

Mitochondrial temperature homeostasis resists external metabolic stresses

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow, and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell-line was estimated to be up to 15 {degrees}C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of OXPHOS complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of oxidative phosphorylation and is homeostatically maintained as a primary feature of mitochondrial metabolism. IMPACT STATEMENTMitochondria are up to 15 {degrees}C hotter than their external environment in living cells. In response to diverse metabolic stresses, mitochondrial temperature re-adjusts to this value whenever possible.

biochemistry↗

bioRxiv2023

Alternative oxidase expression in mtDNA mutator mice improves blood phenotype but enhances inflammatory and stress responses in skeletal muscle

Energetic insufficiency, excess production of reactive oxygen species (ROS) and aberrant signalling partially account for the diverse pathology of mitochondrial diseases. Whether interventions affecting ROS, a regulator of stem-cell pools, could modify somatic stem-cell homeostasis remains unknown. Previous data from mitochondrial DNA (mtDNA) mutator mice showed that increased ROS leads to oxidative damage in erythroid progenitors, causing lifespan-limiting anemia. Also unclear is how ROS-targeted interventions affect terminally differentiated tissues. Here, we set out to test in mtDNA mutator mice how ubiquitous expression of the Ciona intestinalis alternative oxidase (AOX), which attenuates ROS production, affects murine stem-cell pools. We found that AOX does not affect neural stem cells but delays the progression of mutator-driven anemia. Furthermore, when combined with the mutator, AOX potentiates mitochondrial stress and inflammatory responses in skeletal muscle. These differential cell-type-specific findings demonstrate that AOX expression is not a global panacea for the cure of mitochondrial dysfunction. ROS attenuation needs to be carefully studied regarding specific underlying defects before AOX can be safely used in therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/530968v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@17bfd6org.highwire.dtl.DTLVardef@1338f2aorg.highwire.dtl.DTLVardef@1fc269borg.highwire.dtl.DTLVardef@14abc5e_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Nuclear genetic background influences the phenotype of the Drosophila tko25t mitochondrial protein-synthesis mutant

The Drosophila tko25t point mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding, and to vary with mitochondrial genetic background. Here we show similarly profound effects conferred by nuclear genetic background. We backcrossed tko25t into each of two standard nuclear backgrounds, Oregon R and w1118, the latter used as recipient line in many transgenic applications requiring selection for the white minigene marker. In the w1118 background, tko25t flies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygous tko25t females in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years, tko25tflies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in the w1118 background. Phenotypic severity did not correlate with the expression level of the tko gene. Analysis of tko25t hybrids between the two backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal and parent-of-origin dependent determinants. Although some of these effects may be tko25t-specific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of non-lethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.

genetics↗

bioRxiv2022

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the genes numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

systems biology↗

bioRxiv2021

Systemic impact of the expression of the mitochondrial alternative oxidase on Drosophila development

Despite the beneficial effects of xenotopically expressing the mitochondrial alternative oxidase AOX from Ciona intestinalis in mammalian and insect models, important detrimental outcomes have also been reported, raising concerns regarding its potential deployment as a therapeutic enzyme for human mitochondrial diseases. Because of its non-protonmotive terminal oxidase activity, AOX can bypass the cytochrome segment of the respiratory chain whilst not contributing to mitochondrial ATP synthesis. We have previously shown that pupal lethality occurs when AOX-expressing Drosophila larvae are cultured on a low-nutrient diet, indicating that AOX can perturb normal metabolism during development. Here, combined omics analyses revealed multiple correlates of this diet-dependent lethality, including a general alteration of larval amino acid and lipid metabolism, functional and morphological changes to the larval digestive tract, and a drastic decrease in larval biomass accumulation. Pupae at the pre-lethality stage presented a general downregulation of mitochondrial metabolism and a signature of starvation and deregulated signaling. AOX-induced lethality was partially rescued when the low-nutrient diet was supplemented with tryptophan and/or methionine, but not with proline and/or glutamate, strongly suggesting perturbation of one-carbon metabolism. The developmental dependence on tryptophan and/or methionine, associated with elevated levels of lactate dehydrogenase, 2-hydroxyglutarate, choline-containing metabolites and breakdown products of membrane phospholipids, indicates that AOX expression promotes tissue proliferation and larval growth, but this is ultimately limited by energy dissipation due to partial mitochondrial uncoupling. We speculate that the combination of dietary interventions and AOX expression might, nevertheless, be useful for the metabolic regulation of proliferative tissues, such as tumors.

biochemistry↗

bioRxiv2020

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, which in Drosophila melanogaster (Dm) is exceptionally long, 115 amino acids. Molecular modeling predicted this extended linker to fold into a structure similar to the conserved HBD. We measured catalytic activity and substrate binding by EMSA and biolayer interferometry, using a deletion series of protein variants. Both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, whilst loss of the novel HBD led to a [~]90% drop in K[cat] with a decreased K[M], and a large increase in the stability of the RNA/DNA hybrid-enzyme complex. The findings support a bipartite binding model for the enzyme, whereby the second HBD facilitates dissociation of the active site from the product, allowing for processivity. We used shotgun proteomics to identify protein partners of the enzyme involved in mediating these effects. Single-stranded DNA-binding proteins from both the nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, were prominently detected by this method. However, we were not able to document direct interactions between mtSSB and Dm RNase H1 when co-overexpressed in S2 cells, or functional interactions in vitro. Further studies are needed to determine the exact reaction mechanism of Dm RNase H1, the nature of its interaction with mtSSB and the role of the second HBD in both.

molecular biology↗

bioRxiv2019

Minimal effects of spargel (PGC-1α) overexpression in a Drosophila mitochondrial disease model

PGC-1 and its homologues have been proposed to act as master regulators of mitochondrial biogenesis in animals. Most relevant studies have been conducted in mammals, where interpretation is complicated by the fact that there are three partially redundant members of the gene family. In Drosophila, only a single PGC-1 homologue, spargel (srl), is present in the genome. Here we analyzed the effects of srl overexpression on phenotype and on gene expression in tko25t, a recessive bang-sensitive mutant with a global defect in oxidative phosphorylation, resulting in a deficiency of mitochondrial protein synthesis. In contrast to previous reports, we found only minimal effects of substantial overexpression of srl throughout development, on the expression of a representative set of both mtDNA- and nuclear-encoded OXPHOS- related transcripts, both in tko25t mutant flies and heterozygous controls. Sex and genetic background appeared to influence the expression of the tested genes, but srl overexpression or tko25t itself did not have clear-cut or systematic effects thereon. These studies provide no support to the concept of spargel as a global regulator of mitochondrial biogenesis. Summary blurboverexpression of spargel, the fly PGC-1 homologue proposed as a mitochondrial biogenesis regulator, has minimal effects on the phenotype of tko25t, considered a fly model for mitochondrial disease

molecular biology↗