Koroonaviiruse omikrontüve vastane antikehade kaitse ja T-rakuline immuunvastus pärast BNT162b2-vaktsiini tõhustusdoosi saamist

Eesti Arst 2023; 102(2):11

Corrigendum: Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure.

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.</jats:p

Chemical, Physical and Biological Triggers of Evolutionary Conserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

Mitokondriaalse DNA replikatsioon ja rekombinatsioon pärmis

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Mitokondrid on kõrgemate organismide nagu loomade, taimede ja seente rakkudes olevad väikesed organellid. Mitokondrid on esmatähtsad ainevahetusel ja üks nende peamisi ülesandeid on energia tootmine. Selleks on mitokondritel vaja mitmeid valke, mis on koondunud membraaniga seotud kompleksidesse. Enamus nendest valkudest on kodeeritud tuuma kromosoomide poolt, kuid mõned hädavajalikud subühikud mitokondri DNA poolt. Mitokondrid peavad seega oma väikest genoomi hästi säilitama, et vigadeta DNA-d saaks transkribeerida ja funktsionaalseid valke toota, näiteks energia saamiseks. Kaua aega usuti, et kõikide eukarüootsete rakkude mitokondrite DNA näeb samasugune välja ning seda paljundatakse ja säilitatakse sarnaselt nii loomades, taimedes kui seentes. Pärmseeni, mille uurimiseks on olemas head geneetilise vahendid, kasutatakse seepärast eukarüootide mudelina. Selles väitekirjas kirjeldatud analüüsid tõestavad, et kuigi loomade ja pärmide mitokondriaalse DNA (mtDNA) säilitamises on sarnasusi, on seal ka olulisi erinevusi, mida ei ole siiani selgelt välja toodud. Näiteks on loomade mtDNA ringikujuline, pärmide mtDNA aga lineaarne ja harunenud. Loomade DNA süntees algab spetsiaalse RNA-praimeri sünteesiga kindlas DNA punktis. Pärmides paarduvad ja rekombineeruvad homoloogsed DNA järjestused omavahel, mille käigus üks homoloogne DNA invaseerub teise struktuuri. See invasioon tekitab DNA sünteesi alguspunkti, mis funktsionaalselt vastab loomade RNA-praimerile. Need erinevad DNA säilitamise süsteemid vajavad spetsiifilisi valke. Viimastel aastatel on näidatud, et pärmide ja loomade mitokondrites on palju sarnaseid, aga ka mitmeid erinevaid valke. Need leiud ja käesoleva väitekirja tulemused on olulised ka väljaspool baasteadust. Käesolevas uurimuses kasutati mudelorganismina pärmi Candida albicans. Erinevalt enam uuritud pagaripärmist Saccharomyces cerevisiae põhjustab C. albicans naha ja limaskestade nakkusi. Nõrgestunud immuunsüsteemiga patsientidele (nt. vähi ja AIDSi haigetele) võivad need nakkused olla eluohtlikud. Käesoleva töö tulemused võivad aidata leida ravimeid, mis tõrjuvad seenhaigusi, samas inimesele vähe või üldse mitte kahju tegemata.Mitochondria are small separate organelles within cells of higher organisms such as animals, plants or fungi. They are of major importance for the metabolism, and one of their main functions is the production of energy. In order to produce energy, mitochondria need a number of proteins which are grouped to membrane associated complexes to carry out the energy production. Many of these proteins are encoded by genes of the main chromosomes in the nucleus, but a few essential subunits are encoded on DNA within the mitochondria. Mitochondria therefore need to maintain their own small genome with great care to guarantee error-free DNA, that is transcribed and translated in order to deliver functional proteins products e.g. for energy production. It has been a longstanding belief, that mitochondria in different eukaryotic cells carry an equally looking DNA which is reproduced and maintained in the same ways in animals, plants and fungi. Therefore, yeast-fungi are widely used as eukaryotic model organisms because of the availability of genetics tools. The investigations conducted in the course of this thesis show, that although there are indeed similarities between animal and yeast mitochondrial DNA (mtDNA) maintenance, there are also important differences that have not been clearly shown until now. For example, while the mtDNA in animals appears in form of a circular molecule, yeast mitochondria contain linear and branched ones. In animals the DNA reproduction is started by making a specific starter molecule, termed RNA-primer, at a certain point on the DNA. In yeast, homologous DNA sequences are paired and recombined, in the course of which one homologous DNA invades the other. This invasion provides a starting point for DNA synthesis analogous to the RNA-primer that is used in mammals. These differing maintenance systems require specific protein machineries. In recent years it was shown that mitochondria in yeast and animals contain many similar but also a number of differing proteins. Such findings and also the results of this thesis have implications beyond the general use of basic research. The yeast model organism used in experiments of the current work was Candida albicans. Other than the commonly known baker´s yeast Saccharomyces cerevisiae, the potential pathogen C. albicans causes skin and mucosal infections. These can be life threatening for patients whose immune system is compromised (e.g. through cancer or AIDS). Findings like the ones in this thesis open ways to find unique targets for drugs that may specifically attack the pathogen without or little side effects for a patient

Chemical, Physical and Biological Triggers of Evolutionary Sonserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

Chemical, Physical and Biological Triggers of Evolutionary Sonserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness.

Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking

Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness

Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains: number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol traffickin

Early life microbial exposure and fractional exhaled nitric oxide in school-age children: a prospective birth cohort study

Background: Inflammation is a key factor in the pathogenesis of respiratory diseases. Early life exposure to microbial agents may have an effect on the development of the immune system and on respiratory health later in life.In the present work we aimed to evaluate the associations between early life microbial exposures, and fractional exhaled nitric oxide (FeNO) at school age. Methods. Endotoxin, extracellular polysaccharides (EPS) and β(1,3)-D-glucan were measured in living room dust collected at 2-3 months of age in homes of participants of three prospective European birth cohorts (LISA, n = 182; PIAMA, n = 244; and INMA, n = 355). Home dampness and pet ownership were periodically reported by the parents through questionnaires. FeNO was measured at age 8 for PIAMA and at age 10/11 for LISA and INMA. Cohort-specific associations between the indoor microbial exposures and FeNO were evaluated using multivariable regression analyses. Estimates were combined using random-effects meta-analyses. Results: FeNO at school age was lower in children exposed to endotoxin at age 2-3 months (β -0.05, 95% confidence interval (CI) -0.10;-0.01) and in children with reported dog ownership during the first two years of life (GM ratio 0.82, CI 0.70-0.96). FeNO was not significantly associated with early life exposure to EPS, β(1,3)-D-glucan, indoor dampness and cat ownership. Conclusion: Early life exposure to bacterial endotoxin and early life dog ownership are associated with lower FeNO at school age. Further studies are needed to confirm our results and to unravel the under