Papers by Svetlana Konovalova
bioRxiv (Cold Spring Harbor Laboratory), Apr 11, 2024
Peroxisomes are single membrane-bound organelles essential for human health, yet the mechanism of... more Peroxisomes are single membrane-bound organelles essential for human health, yet the mechanism of peroxisomal biogenesis remains poorly described. Here using a systematic double screening approach, we identified ribosome-binding protein 1, RRBP1, as a novel peroxisomal biogenesis factor in human cells. We showed that the number of peroxisomes and the levels of peroxisomal proteins are reduced by RRBP1 KO in HEK293T cells. In addition, the processing of peroxisomal matrix proteins, such as ACOX1 and thiolase was affected in RRBP1 KO HEK293T cells. Using electron microscopy, we showed that RRBP1, an ER membrane protein, is involved in the maintaining of the long-distance contact sites between ER and peroxisomal membranes, suggesting that the role of RRBP1 in peroxisomal biogenesis is mediated through inter-organellar contact sites. Altogether our results showed that RRBP1 promotes peroxisomal biogenesis in human cells highlighting the power of systematic approaches in discovering novel factors of organellar biogenesis.
PLOS ONE, Nov 30, 2023
Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile a... more Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.
European Journal of Medical Genetics, Mar 1, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Toxicology in Vitro, Apr 1, 2016
The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mit... more The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4′diisothiocyanostilbene-2,2′-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO 3 and KNO 3 stimulated the Tl + -induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨ mito ), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl + -induced MPTP in the inner membrane of Ca 2+ -loaded rat liver mitochondria. Using the Tl + -induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.
Proceedings of the National Academy of Sciences
Cardiolipin (CL) is an essential phospholipid for mitochondrial structure and function. Here, we ... more Cardiolipin (CL) is an essential phospholipid for mitochondrial structure and function. Here, we present a small mitochondrial protein, NERCLIN, as a negative regulator of CL homeostasis and mitochondrial ultrastructure. Primate-specific NERCLIN is expressed ubiquitously from the GRPEL2 locus on a tightly regulated low level. NERCLIN overexpression severely disrupts mitochondrial cristae structure and induces mitochondrial fragmentation. Proximity labeling and immunoprecipitation analysis suggested interactions of NERCLIN with CL synthesis and prohibitin complexes on the matrix side of the inner mitochondrial membrane. Lipid analysis indicated that NERCLIN regulates mitochondrial CL content. Furthermore, NERCLIN is responsive to heat stress ensuring OPA1 processing and cell survival. Thus, we propose that NERCLIN contributes to the stress-induced adaptation of mitochondrial dynamics. Our findings add NERCLIN to the group of recently identified small mitochondrial proteins with impor...
ABSTRACTPeroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown... more ABSTRACTPeroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, glycolysis, and biosynthesis of sterols and ether lipids. Defects in peroxisome biogenesis result in severe neurological diseases, such as Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and myelopathies. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter ER in both WT and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. However, in PEX3 KO cells, which lack peroxisomes as well, the endogenous TA protein, ACBD5, does not target mitochondria, suggesting that PEX3 plays an important role in targeting of peroxisomal TA proteins to mitochondria. Final...
Current Protocols in Toxicology, 2018
Mitochondria are multifunctional organelles with their own genome and protein synthesis machinery... more Mitochondria are multifunctional organelles with their own genome and protein synthesis machinery. The 13 proteins encoded by mitochondrial DNA (mtDNA) are core subunits of the oxidative phosphorylation (OXPHOS) system producing the majority of cellular ATP. Yet most mitochondrial proteins are encoded by nuclear genes, synthesized by cytosolic ribosomes, and imported into mitochondria. Therefore, disturbances in cytosolic proteostasis have consequences on the gene expression and synthesis of mtDNA‐encoded proteins and overall on mitochondrial function. Internal and environmental factors such as mutations, aging, oxidative stress, and toxic agents can affect the translation and the stability of mitochondrial proteins and lead to OXPHOS dysfunction. Here, methods for analysis of mitochondrial translation rate and protein stability using radioactive and non‐radioactive technique as well as the methods for studying steady‐state levels and assembly of OXPHOS complexes are described. © 20...
Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in diseases affecting mo... more Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in diseases affecting motor neurons such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease, and form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Knockout iPSC and motor neurons sustain mitochondrial ultrastructure and show reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that absence of CHCHD2 or CHCHD10 does not disrupt functionality, but induces similar modifications in human motor neurons. Thus pat...
European Journal of Medical Genetics, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nucleic Acids Research, 2017
Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by ami... more Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.
Human molecular genetics, Jan 4, 2018
Recessively-inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA syntheta... more Recessively-inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase (COX)-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequ...
Toxicology in Vitro, 2016
The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mit... more The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4′diisothiocyanostilbene-2,2′-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO 3 and KNO 3 stimulated the Tl + -induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨ mito ), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl + -induced MPTP in the inner membrane of Ca 2+ -loaded rat liver mitochondria. Using the Tl + -induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.
BBA Clinical, 2015
Background: HSPB1 belongs to the family of small heat shock proteins (sHSP) that have importance ... more Background: HSPB1 belongs to the family of small heat shock proteins (sHSP) that have importance in protection against unfolded protein stress, in cancer cells for escaping drug toxicity stress and in neurons for suppression of protein aggregates. sHSPs have a conserved α-crystalline domain (ACD), flanked by variable N-and C-termini, whose functions are not fully understood. Dominant missense variants in HSPB1, locating mostly to the ACD, have been linked to inherited neuropathy. Methods: Patients underwent detailed clinical and neurophysiologic characterization. Disease causing variants were identified by exome or gene panel sequencing. Primary patient fibroblasts were used to investigate the effects of the dominant defective HSPB1 proteins. Results: Frameshift variant predicting ablation of the entire C-terminus p.(Met169Cfs2*) of HSPB1 and a missense variant p.(Arg127Leu) were identified in patients with dominantly inherited motor-predominant axonal Charcot-Marie-Tooth neuropathy. We show that the truncated protein is stable and binds wild type HSPB1. Both mutations impaired the heat stress tolerance of the fibroblasts. This effect was particularly pronounced for the cells with the truncating variant, independent of heat-induced nuclear translocation and induction of global transcriptional heat response. Furthermore, the truncated HSPB1 increased cellular sensitivity to protein misfolding. Conclusion: Our results suggest that truncation of the non-conserved C-terminus impairs the function of HSPB1 in cellular stress response. General significance: sHSPs have important roles in prevention of protein aggregates that induce toxicity. We showed that C-terminal part of HSPB1 is critical for tolerance of unfolded protein stress, and when lacking causes axonal neuropathy in patients.
Mitochondrion, 2012
The effect of pinacidil was studied on calcium-loaded rat heart mitochondria (RHM) in the presenc... more The effect of pinacidil was studied on calcium-loaded rat heart mitochondria (RHM) in the presence of succinate and rotenone. In experiments with pinacidil, the swelling of these mitochondria increased in media with NH 4 NO 3 or K-acetate, but the inner membrane potential ΔΨ mito and state 3 or 2,4-dinitrophenol-uncoupled respiration of these organelles were decreased due to opening of the mitochondrial permeability transition pore in the inner membrane. These effects were inhibited by cyclosporin A and ADP. It was concluded that the protective effect of pinacidil in the cardiac muscle ischemia/reperfusion may be associated with stimulation mitochondrial swelling and a decrease in RHM calcium overload resulted in a decrease in ΔΨ mito due to the soft uncoupling pinacidil effect.
European Journal of Human Genetics, 2013
Charcot-Marie-Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantl... more Charcot-Marie-Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantly inherited axonal CMT2 displays striking genetic heterogeneity, with 17 presently known disease genes. The large number of candidate genes, combined with lack of genotype-phenotype correlations, has made genetic diagnosis in CMT2 time-consuming and costly. In Finland, 25% of dominant CMT2 is explained by either a GDAP1 founder mutation or private MFN2 mutations but the rest of the families have remained without molecular diagnosis. Whole-exome and genome sequencing are powerful techniques to find disease mutations for CMT patients but they require large amounts of sequencing to confidently exclude heterozygous variants in all candidate genes, and they generate a vast amount of irrelevant data for diagnostic needs. Here we tested a targeted nextgeneration sequencing approach to screen the CMT2 genes. In total, 15 unrelated patients from dominant CMT2 families from Finland, in whom MFN2 and GDAP1 mutations had been excluded, participated in the study. The targeted approach produced sufficient sequence coverage for 95% of the 309 targeted exons, the rest we excluded by Sanger sequencing. Unexpectedly, the screen revealed a disease mutation only in one family, in the HSPB1 gene. Thus, new disease genes underlie CMT2 in the remaining families, indicating further genetic heterogeneity. We conclude that targeted next-generation sequencing is an efficient tool for genetic screening in CMT2 that also aids in the selection of patients for genome-wide approaches.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
eg. Fura-2). Here, we report a red-shifted ATeam (GO-ATeam) that has GFP and OFP as a donor and a... more eg. Fura-2). Here, we report a red-shifted ATeam (GO-ATeam) that has GFP and OFP as a donor and an acceptor of FRET, respectively, instead of CFP and YFP. Because GO-ATeam is excited with longer wavelength light than the previous ATeam, it is compatible with a UV-excitable ratiometric calcium indicator, Fura-2. We simultaneously imaged ATP and Ca 2+ concentrations of the same single living cells by loading Fura-2 calcium indicator into the cells expressing GO-ATeam. When Ca 2+ spark was induced in the cells by histamine, mitochondrial also ATP elevated following Ca 2+ . Intracellular Ca 2+ typically returned to a basal level within 10 minutes after histamine stimulation. In contrast, most of cells retained high level of mitochondrial ATP for a much longer time (sometimes more than 30 min). This result strongly supports the idea that intracellular Ca 2+ promotes long-term activation of energy metabolism in mitochondria [1].
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
exercise training improves heart function and cardiovascular disease risk profile are not underst... more exercise training improves heart function and cardiovascular disease risk profile are not understood. The purpose of the present investigation was to study the effect of long-term exercise training on the sensitivity of calcium-induced MPTP opening and the mitochondrial uncoupling protein 3 (UCP3) expression. The animals were divided into 2 groups: 1st groupintact adult animals (control rats), 2nd groupadult animals, which are subject to dosed physical load carried by the forced swimming five days a week for six weeks (trained rats). It was shown that the sensitivity of MPTP-opening to Ca 2+ in the trained rats heart decreased compared with control animals. Thus, in the trained rats heart mitochondria had a significant increase cNOS activity almost twice, slight increase in the NOS activity compared with control, and slight increasing the hydrogen peroxide. Тhe expression level of UCP3 was reduced by 65% in heart mitochondria of long-term exercise training rat compared with the control. These results suggest that decreased the expression level of UCP3 in heart mitochondria of the trained rats may play the certain role in the complex mechanism of adaptive response of the heart, that may be aimed mainly on the efficiency of oxidative phosphorylation in mitochondria and, consequently, to increase the synthesis ATP. Thus, long-term exercise training contribute tо reducing the sensitivity of the MPTP opening to the action of calcium ions by increasing the activity of mitochondrial constitutive NOS and synthesis of nitric oxidean endogenous inhibitor of MPTP opening.
Journal of Visualized Experiments
Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mit... more Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.
Neurobiology of Disease, 2020
Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases... more Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.
The International Journal of Biochemistry & Cell Biology, 2015
Impairment of mitochondrial protein homeostasis disrupts mitochondrial function and causes human ... more Impairment of mitochondrial protein homeostasis disrupts mitochondrial function and causes human diseases and aging, but the molecular mechanisms of protein synthesis and quality control in mammalian mitochondria are not fully understood. Here we demonstrate in human cells that misincorporation of an arginine analog, canavanine, during mitochondrial protein synthesis, induced aberrant translation products and destabilized the mtDNA-encoded proteome, leading to loss of mitochondrial respiratory chain complexes. Furthermore, in the presence of a high concentration of canavanine, mitoribosome stalling could be demonstrated. The stalling did not, however, occur at arginine codons, but downstream of those codons. In particular, two adjacent arginines induced the most prominent downstream stalling effect, with the distance between the arginine codons and the stalling peak corresponding roughly to the length of the ribosomal exit tunnel. These results suggest that misincorporated canavanine disrupted the proper folding of the hydrophobic nascent polypeptides within the exit tunnel or while being inserted into the inner mitochondrial membrane. The canavanine treatment provides a model system for studying the consequences of mitoribosome stalling and the responses to misfolded proteins exiting the mitochondrial ribosome.
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Papers by Svetlana Konovalova