Biology subjects

Pines, O.

Publications and source records attributed to Pines, O..

6 recordsLinked to original sources

bioRxiv2025

Succinate and its carrier Sfc1 mediate metabolic control of mitochondrial protein import by the TIM23 translocase

Tim23 is an essential component of the mitochondrial inner membrane translocase and Sfc1 is a carrier that exchanges succinate for fumarate across that membrane. Sfc1 and succinic acid availability regulate dual targeting of fumarase and aconitase by facilitating mitochondrial import of their newly synthesized precursors, as shown by pulse-chase experiments. Here we show that Sfc1 directly interacts with the Tim23 and succinate affects this interaction, which in turn affects mitochondrial protein import. Physical interaction between Tim23 and Sfc1 was proven by co immunoprecipitation, Bimolecular Fluorescence Complementation (BiFC) and Biotin-based proximity labeling (TurboID). Proximity labeling-informed mutagenesis allowed us to dissect the carrier activity of Sfc1 from its function as a TIM23 regulator. We performed Rosetta-MP docking of Sfc1 and Tim23 to envisage the interface. Thus, our findings show that metabolites can regulate mitochondrial import and adjust the segregation of key metabolic enzymes between the cytosol and mitochondria.

cell biology↗

bioRxiv2025

Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells.

Mutations in the mitochondrial DNA (mtDNA) are associated with severe human diseases, lacking efficient therapies. Direct correction of mtDNA mutations may offer a cure for such diseases. We propose a novel strategy based on double-stranded DNA (dsDNA) oligonucleotide delivery into mitochondria and intrinsic microhomology-mediated end joining (MMEJ) for mtDNA editing. This strategy enables introduction of multiple predefined nucleotide changes in mtDNA. For this, the presence of MMEJ activity in the human mitochondrial lysates was confirmed. 49 bp DNA oligonucleotide duplexes, fused to an RNA hairpin previously identified as a mitochondrial import signal, were delivered into the mitochondria of cultured human cells. Delivery of these donor dsDNA molecules, homological to an ND4 site of mtDNA and bearing designed nucleotide changes, led to a low but statistically significant introduction of the designed nucleotide changes into mtDNA. Donor dsDNA delivery combined with the CRISPR/mito-AsCas12a system also resulted in a statistically significant number of an expected concomitant change of five nucleotides distributed across a 16-nucleotide ND4 site of the mitochondrial genome. The proposed strategy may become an efficient mtDNA editing tool suitable for the correction of near-homoplasmic mutations such as Lebers Hereditary Optic Neuropathy (LHON)-associated mutations in the ND4 gene of mtDNA.

molecular biology↗

bioRxiv2022

Yeast Mitochondrial Derived Vesicles (MDVs) have membrane potential and contain a functional ATP synthase

Vesicular transport is a means of communication. While cells can communicate between each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, in particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here we show that functional mitochondria, isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We were then able for the first time to isolate these Mitochondrial Derived Vesicles (MDVs) and found that they are relatively uniform in size, of about 100nm and carry selective protein cargo including enrichment of ATP synthase subunits. Remarkably, we further found that these MDVs harbor a functional ATP synthase complex. Moreover, we demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP producing vesicles, which can potentially regenerate ATP deficient mitochondria and may participate in organelle to organelle communication.

cell biology↗

bioRxiv2022

Depletion of FH, an essential TCA cycle enzyme, drives proliferation in a two-step model

Several tumor suppressor genes do not follow the canonical function of cell cycle repressors. For example, fumarate hydratase (FH) is an evolutionary conserved TCA cycle enzyme that reversibly catalyzes the hydration of fumarate to L-malate and has a moonlight function in the DNA damage response (DDR). Interestingly, despite this enzymes essential role in central carbon metabolism, FH is inactive or absent in several tumors, such as hereditary leiomyomatosis and renal cell cancer (HLRCC). Accordingly, FH has a contradictory cellular function, as it is pro-survival through its role in the TCA cycle, yet its loss can drive tumorigenesis. These observations have supported the role of FH as a tumor suppressor. Here, we solved this contradiction by determining the molecular mechanisms that allow the cells to survive and even proliferate upon FH loss. We found that the cells response to FH loss is separated into two distinct time frames based on cell proliferation and DNA damage repair. During the early stages of FH loss, the cells proliferation and DNA damage repair are inhibited. However, over time the cells overcome the FH loss and form knockout clones, indistinguishable from WT cells in their proliferation rate. Due to the FH loss effect on DNA damage repair, we assumed that the recovered cells bear adaptive mutations. Therefore, we applied whole-exome sequencing to identify such mutated genes systematically. Indeed, we identified recurring mutations in genes belonging to the central oncogenic signaling pathways, such as JAK/STAT, which we validated to be impaired in FH-KO clones. Intriguingly, we demonstrated that these adaptive mutations are responsible for FH-KO cell proliferation under TCA cycle malfunction.

cancer biology↗

bioRxiv2022

Ubiquitination occurs in the mitochondrial matrix byeclipsed targeted components of the ubiquitin machinery

Ubiquitination is a critical type of post translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent -complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Subsequently, the results with respect to MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity in the cytosol/nucleus. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective of mitochondrial and ubiquitination research.

cell biology↗

bioRxiv2020

Class-I and Class-II fumarases are a paradigm of the recruitment of metabolites and metabolic enzymes for signalling of the DNA Damage Response during evolution

Class-II fumarases (Fumarate Hydratase, FH) are dual targeted enzymes, occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and more specifically, protecting cells from DNA double strand breaks. Similarly, the Gram-positive Bacterium Bacillus subtilis Class-II fumarase, in addition to its role in the TCA cycle, also participates in the DDR. Escherichia coli, harbors three fumarase genes; Class-I fumA and fumB and Class-II fumC. Notably, Class-I fumarases, show no sequence similarity to Class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between Class-I fumarases which participate in the DDR, and the Class-II fumarase which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (-KG), has a novel function, complementing DNA damage sensitivity of fum null mutants. Excitingly, we identify the E. coli -KG dependent DNA repair enzyme AlkB, as the target of this interplay of metabolite signaling. In addition to -KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering -KG). The second is a more global effect on transcription, as fum null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme Class preforming the function. Significance StatementClass-II fumarases have been shown to participate in cellular respiration and the DNA damage response. Here we show, for the first time, that in the model prokaryote, Escherichia coli, which harbors both Class-I and Class-II fumarases, it is the Class-I fumarases that participate in DNA damage repair by a mechanism which is different than those described for other fumarases. Strikingly, this mechanism employs a novel signaling molecule, alpha-ketoglutarate (-KG), and its target is the DNA damage repair enzyme AlkB. In addition, we show that fumarase precursor metabolites, fumarate and succinate, can inhibit the -KG-dependent DNA damage repair enzyme, AlkB, both in vitro and in vivo. This study provides a new perspective on the function and evolution of metabolic signaling.

microbiology↗