Biology subjects

Filipovic, M.

Publications and source records attributed to Filipovic, M..

8 recordsLinked to original sources

bioRxiv2026

Adulthood depletion of Integrator extends lifespan and healthspan via defective pre-mRNA processing

Identifying strategies to mitigate age-related physiological decline remains a central challenge. During ageing, the transcriptome undergoes extensive remodelling, but how this affects organismal health and lifespan is not well understood. The Integrator complex plays a central role in RNA polymerase II transcription and RNA 3' end processing. Surprisingly, we find that depletion of most Integrator subunits specifically in adults extends lifespan and healthspan in the nematode C. elegans. We show that loss of the catalytic subunit INTS-11 disrupts 3' end formation of small nuclear and spliced leader RNAs, impairing trans-splicing and promoting outron retention in a subset of transcripts enriched for spliceosomal and nucleocytoplasmic transport genes. These RNA-processing defects lead to altered levels of endogenous siRNAs, which are required for the longevity and healthspan benefits of INTS-11 depletion. In parallel, outron retention disrupts nuclear-encoded mitochondrial gene expression and protein production, inducing mitochondrial dysfunction and promoting lifespan extension. We also demonstrate that loss of INTS-11 perturbs transcription elongation at genes where Integrator is present at promoters, and that upregulation of enhancer elements within intragenic regions can affect the expression and isoform usage of nearby genes. Together, our findings identify Integrator as a key upstream regulator of non-coding RNA transcription, which in turn impacts protein-coding gene expression and mitochondrial function to shape the ageing process.

cell biology↗

bioRxiv2025

The transsulfuration pathway suppresses the embryonic lethalphenotype of glutathione reductase mutants in Caenorhabditis elegans

The gsr-1 gene encodes the only glutathione reductase in Caenorhabditis elegans and gsr-1 loss of function alleles have a fully penetrant embryonic lethal phenotype. Therefore, maintenance of glutathione redox homeostasis is essential for nematode survival. We report here that impairment of the nonsense-mediated mRNA decay (NMD) pathway suppresses the embryonic lethality of gsr-1 mutants, allowing their normal development and growth. This NMD pathway dependent suppression requires cth-1 and cth-2 that encode, respectively, two isoforms of cystathionine-{gamma}-lyase that catalyze the conversion of cystathionine to cysteine through the transsulfuration pathway. Interestingly, the thioredoxin system that can also provide cysteine through the cystine reduction pathway is not required for the suppression of the lethal phenotype of gsr-1 embryos when the NMD pathway is inactivated. Together, our data indicate that increasing the activity of the reverse transsulfuration pathway can compensate the detrimental effect of the gsr-1 mutation, raising the interesting question of why C. elegans has not preserved such compensatory mechanism to avoid the embryonic lethality of these mutants. HighlightsO_LIA novel cryptic deletion in the smg-3 gene suppresses the embryonic lethality of C. elegans gsr-1 mutants. C_LIO_LIInactivation of the nonsense-mediated mRNA decay (NMD) pathway allows survival of gsr-1 mutant worms. C_LIO_LIGenetic impairment of the transsulfuration pathway restores the embryonic lethal phenotype in gsr-1; smg-3 mutants. C_LI

genetics↗

bioRxiv2024

Endogenously produced hydrogen cyanide serves as a novel mammalian gasotransmitter

Small, gaseous molecules, known as gasotransmitters (NO, CO, H2S), are produced endogenously in mammalian cells and serve important biological roles. Hydrogen cyanide, traditionally considered a cytotoxic molecule in mammals, serves as an endogenous mediator in several plants and bacterial species. Here we show that low concentrations of cyanide are generated endogenously in mouse liver and human hepatocytes. Cyanide production is stimulated by glycine, occurs at the low pH of lysosomes and requires peroxidase activity. Cyanide, in turn, is detectable in several cellular compartments. Cyanide is also detectable basally in the blood of mice; its levels increase after treatment of the animals with glycine. Rhodanese activity regulates endogenous cyanide levels. Cyanide, when generated endogenously at an optimal level, exerts stimulatory effects on mitochondrial bioenergetics, cell metabolism and cell proliferation. Dysregulation of endogenous cyanide, either below or above optimal levels, impairs cellular bioenergetics. The regulatory effects of cyanide are in part mediated by posttranslational modification of cysteine residues via protein cyanylation; cyanylated protein residues can be detected basally, and increase after treatment with glycine. Controlled low-dose cyanide supplementation exhibits cytoprotective effects, as demonstrated in hypoxia and reoxygenation models in vitro and in vivo. However, pathologically elevated cyanide production, as demonstrated in nonketotic hyperglycinemia - an autosomal recessive disease of glycine metabolism - is deleterious to the cells.

biochemistry↗

bioRxiv2024

Sulfite oxidase deficiency causes persulfidation loss and H2S release

Sulfite oxidase (SOX) deficiency is a rare inborn error of cysteine metabolism resulting in severe neurological damage. In patients, sulfite accumulates to toxic levels causing a raise in downstream products S-sulfocysteine (SSC), mediating excitotoxicity, and thiosulfate, a catabolic intermediate/product of H2S metabolism. Here, we report a full-body knock-out mouse model for SOX deficiency (SOXD) with a severely impaired phenotype. Amongst the urinary biomarkers, thiosulfate showed a 45-fold accumulation in SOXD mice representing the major excreted S-metabolite. Consistently, we found increased plasma H2S, which was derived from sulfite-induced release from persulfides as demonstrated in vitro and in vivo. Mass spectrometric analysis of total protein persulfidome identified a major loss of persulfidation in 20% of the proteome affecting enzymes in amino acids and fatty acid metabolism. Urinary amino acid profiles indicate metabolic rewiring suggesting partial reversal of the TCA cycle thus identifying a novel contribution of H2S metabolism and persulfidation in SOXD.

biochemistry↗

bioRxiv2023

Protein thiol alterations drive aberrant phase separation in aging

Cellular homeostasis relies on precise regulation through chemical processes, such as protein posttranslational modifications (PTM) and physical processes, such as biomolecular condensation. Aging disrupts this balance, increasing susceptibility to diseases and death. However, the mechanisms behind age-related pathogenesis remain elusive. In this study, we dissected various cysteine PTMs and their impact on protein-mediated biomolecular condensation in aging brain. Our findings reveal that aging is associated with significant remodeling of cysteine PTMs, which impacts protein ability to participate in liquid-liquid phase separation (LLPS). Specifically, aging leads to an increase in protein sulfenylation and sulfonylation, which promotes LLPS and through conformational change increases the propensity of proteins to aggregate. Protein persulfidation, a protective thiol modification, prevents this by causing condensate dissolution. We demonstrate that age-induced alterations in cysteine PTMs influence the LLPS properties of synapsin-1 and G3BP2, resulting in disruptions in neurotransmitter release and stress granule formation, respectively. Additionally, our study uncovers that GAPDH is susceptible to LLPS and cysteine sulfonylation exacerbates its transition from condensates to aggregates. Mice deficient in cystathionine gamma-lyase, a pro-longevity gene that regulates intracellular persulfide levels, exhibit a shorter lifespan and spontaneous development of neurofibrillary tangles.

biochemistry↗

bioRxiv2021

The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain

The human TRPA1 (hTRPA1) is an intrinsic thermosensitive ion channel responding to both cold and heat, depending on the redox environment. Here, we have studied purified hTRPA1 truncated proteins to gain further insight into the temperature gating of hTRPA1. We found in patch-clamp bilayer recordings that {Delta}1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas {Delta}1-854 hTRPA1 that is also lacking the S1-S4 voltage sensing-like domain (VSLD) gained sensitivity to cold but lost its heat sensitivity. The thiol reducing agent TCEP abolished the temperature sensitivity of both {Delta}1-688 hTRPA1 and {Delta}1-854 hTRPA1. Cold and heat activity of {Delta}1-688 hTRPA1 and {Delta}1-854 hTRPA1 were associated with different structural conformational changes as revealed by intrinsic tryptophan fluorescence measurements. Heat evoked major structural rearrangement of the VSLD as well as the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD), whereas cold only caused minor conformational changes. As shown for {Delta}1-854 hTRPA1, a sudden drop in tryptophan fluorescence occurred within 25-20{degrees}C indicating a transition between heat and cold conformations of the CTD, and thus it is proposed that the CTD contains a bidirectional temperature switch priming hTRPA1 for either cold or heat. In whole-cell patch clamp electrophysiology experiments, replacement of the cysteines 865, 1021 and 1025 with alanine modulated the cold sensitivity of hTRPA1 when heterologously expressed in HEK293T cells. It is proposed that the hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

physiology↗

bioRxiv2020

ATF-4 and hydrogen sulfide signalling mediate longevity from inhibition of translation or mTORC1

Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H2S) production. This H2S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H2S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H2S levels, or enhancing mechanisms that H2S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition.

physiology↗

bioRxiv2019

Direct Pathway Neurons in Mouse Dorsolateral Striatum In Vivo Receive Stronger Synaptic Input than Indirect Pathway Neurons

Striatal projection neurons, the medium spiny neurons (MSNs), play a crucial role in various motor and cognitive functions. MSNs express either D1 or D2 type dopamine receptors and initiate the direct-pathway (dMSNs) or indirect pathways (iMSNs) of the basal ganglia, respectively. dMSNs have been shown to receive more inhibition than iMSNs from intrastriatal sources. Based on these findings, computational modelling of the striatal network has predicted that under healthy conditions dMSNs should receive more excitatory input than iMSNs. To test this prediction, we analyzed in vivo whole-cell recordings from dMSNs and iMSNs in healthy and dopamine-depleted (6OHDA) anaesthetized mice. By comparing their membrane potential fluctuations, we found that dMSNs exhibited considerably larger membrane potential fluctuations over a wide frequency range. Furthermore, by comparing the spike-triggered average membrane potentials, we found that dMSNs depolarized towards the spike threshold significantly faster than iMSNs did. Together, these finding corroborate the theoretical prediction that direct-pathway MSNs receive stronger input than indirect-pathway neurons. Finally, we found that dopamine-depleted mice exhibited no difference between the membrane potential fluctuations of dMSNs and iMSNs. These data provide new insights into the question how a lack of dopamine may lead to behavior deficits associated with Parkinsons disease.\n\nSignificance statementThe direct and indirect pathways of the basal ganglia originate from the D1 and D2 type dopamine receptor expressing medium spiny neurons (dMSNs and iMSNs), respectively. To understand the role of the striatum in brain function and dysfunction it is important to characterize the differences in synaptic inputs to the two MSN types. Theoretical results predicted that dMSNs should receive stronger excitatory input than iMSNs. Here, we studied membrane potential fluctuation statistics of MSNs recorded in vivo in anaesthetized mice and found that dMSNs, indeed, received stronger synaptic input than iMSNs. We corroborated this finding by spike-triggered membrane potential analysis, showing that dMSNs spiking required more synaptic input than iMSNs spiking did, as had been predicted by computational models.

neuroscience↗