Biology subjects

Atanassov, I.

Publications and source records attributed to Atanassov, I..

7 recordsLinked to original sources

bioRxiv2026

Post-assembly proteostasis of the respiratory chain is independent of respirasome organization

Respirasomes have long been proposed to stabilize their constituent complexes. Yet the close relationship between individual complex biogenesis and respirasome formation has made it difficult to determine whether respirasomes are required for complex maintenance after assembly. Here, we uncoupled these processes using a genetic perturbation of the CI-CIII2 contact site that severely depletes respirasomes while preserving individual complex assembly and function. Across orthogonal measures of protein stability, turnover and solubility, respiratory-chain maintenance remained largely preserved despite severe respirasome depletion in mouse embryonic fibroblasts, even under mitochondrial stress. We further tested whether a requirement for respirasomes emerges in vivo when OXPHOS biogenesis is progressively impaired through tissue-specific TFAM deletion. Respirasome depletion did not accelerate the loss of CI, CIII2 or CIV during disease progression. Together, our findings show that respirasomes are not a major determinant of post-assembly respiratory-chain complex maintenance, even when mitochondrial homeostasis and OXPHOS biogenesis are challenged.

cell biology↗

bioRxiv2024

The mitochondrial methylation potential gates mitoribosome assembly

S-adenosylmethionine (SAM) is crucial for cellular processes, primarily serving as the principal methyl group donor of the cell and playing a key role in gene regulation and translation on the ribosome. Inside mitochondria, SAM-dependent methylations occur at several steps of gene expression, but their role and significance remain unclear. Using direct long-read RNA sequencing on mouse tissue and mouse embryonic fibroblasts, we demonstrate that the mitochondrial ribosomal gene cluster is not efficiently processed without mitochondrial SAM. This results in the accumulation of unprocessed ribosomal RNA precursors. Protein profiling of ribosome fractions revealed that these precursors are associated with processing and ribosome assembly factors, indicating stalling at an early stage. Structural analysis of the mitochondrial ribosome by cryogenic electron microscopy revealed that mitochondrial SAM is required during peptidyl transferase centre formation and mitochondrial ribosome assembly. Our data thus identify a critical role for methylation at two steps during mitochondrial gene expression.

molecular biology↗

bioRxiv2023

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III2 and IV (CI, CIII2 and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI- CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knock-in mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in the mouse, but raises questions about their alternate functions, such as relating to regulation of protein stability and prevention of age-associated protein aggregation.

molecular biology↗

bioRxiv2023

Hexokinase regulates Mondo-mediated longevity via the PPP and organellar dynamics

The transcriptional complex Mondo/Max-like, MML-1/MXL-2, acts as a convergent transcriptional regulatory output of multiple longevity pathways in Caenorhabditis elegans. These transcription factors coordinate nutrient sensing with carbohydrate and lipid metabolism across the evolutionary spectrum. While most studies have focused on the downstream outputs, little is known about the upstream inputs that regulate these transcription factors in a live organism. Here, we found that knockdown of various glucose metabolic enzymes decreases MML-1 localization in the nucleus and identified two hexokinase isozymes, hxk-1 and hxk-2, as the most vigorous regulators of MML-1 function. Upon hexokinase knockdown, MML-1 redistributes to mitochondria and lipid droplets (LD), and concomitantly, transcriptional targets are downregulated and germline longevity is abolished. Further, we found that hxk-1 regulates MML-1 through mitochondrial {beta}-oxidation, while hxk-2 regulates MML-1 by modulating the pentose phosphate pathway (PPP) and its coordinated association with lipid droplets. Similarly, inhibition of the PPP rescues mammalian MondoA nuclear translocation and transcriptional function upon starvation. These studies reveal how metabolic signals and organellar communication regulate a key convergent metabolic transcription factor to promote longevity.

cell biology↗

bioRxiv2022

Metabolic rewiring of mitochondria in senescence revealed by time-resolved analysis of the mitochondrial proteome

Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early-responding pathways, the degradation of branched-chain amino acid (BCAA) was increased, while the one carbon-folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic tracing experiments, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide an unprecedentedly comprehensive view on the metabolic status of mitochondria in senescent cells and reveal how the mitochondrial proteome adapts to the induction of senescence.

cell biology↗

bioRxiv2020

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response, is implicated in cancer and diabetes, and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the first and rate-limiting HP enzyme. GFAT-1 activity is modulated through UDP-GlcNAc feedback inhibition and by kinase signaling, including Ser205 phosphorylation by protein kinase A (PKA). The consequence and molecular mechanism of GFAT-1 phosphorylation, however, remains poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans, leading to ER stress resistance. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and PKA-mediated Ser205 phosphorylation. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, GFAT-1 phosphorylation by PKA uncoupled the feedback loop of the HP and, depending on UDP-GlcNAc availability, phosphorylation by PKA results in lower or enhanced GFAT-1 activity in vivo. Mechanistically, our data indicate that the relative positioning of the two GFAT-1 domains might be affected by phosphorylation and we propose a model how Ser205 phosphorylation modulates the activity and feedback inhibition of GFAT-1.

biochemistry↗

bioRxiv2019

Versatile proteome labelling in fruit flies with SILAF

Drosophila melanogaster has been a working horse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to technical obstacles, especially the lack of reliable labelling methods. Here, we advanced a chemically defined food source into stable-isotope labelling of amino acids in flies (SILAF). It allows for the rapid generation of a large number of flies with full incorporation of lysine-6. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 5,966 proteins and 7,496 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. Furthermore, the label can be traced and predicts protein turnover rates during different developmental stages. The ease and versatility of the method actuates the fruit fly as an appealing model in proteomic and post-translational modification studies and it enlarges potential metabolic applications based on heavy amino acid diets.

molecular biology↗