Mitokondriaalsete nukleoidide ja mitokondritega seotud ER-membraanide (MAM) koosmõju inimrakkudes

Mitokondriaalne DNA (mtDNA) moodustab rakkudes nukleoproteiinkomplekse, mida nimetatakse nukleoidideks. MtDNA säilitamine on raku elutegevuse seisukohalt oluline, kuna häired mtDNA-s põhjustavad haiguseid. Mitokondriaalsed nukleoidid assotsieeruvad mitokondri sisemembraaniga ning kolesteroolirikka mikrodomääniga, mis tõenäoliselt koordineerivad mtDNA säilitamist. Mitokondrid on kontaktis ER membraanidega (MAM), mille kaudu reguleeritakse arvatavasti ka mtDNA säilitamist/segregatsiooni. Käesolevas töös uuriti nukleoidide võimalikke mõjusid MAM-idele ning MAM-ide võimalikke mõjusid nukleoididele kasutades siRNA vahendatud knockdown meetodit. Selgus, et nukleoidi valk TFAM mõjutab selgelt nii nukleoide kui MAM-struktuure. MAM-ide valk Mfn2 ei avaldanud tugevat mõju nukleoididele. Nukleoididega seotud ATAD3 mõjutas sarnaselt TFAM mahasurumisele MAM-struktuure

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.publishedVersionPeer reviewe

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

Inimese mitokondriaalsete nukleoidide dünaamika mitokondriaalses võrgustikus

Väitekirja elektrooniline versioon ei sisalda publikatsiooneMitokondrid on endosmbiontset päritolu päristuumse raku organellid, millede enimtuntud funktsiooniks on makroergilise ühendi ATP tootmine oksüdatiivse fosforüülimise teel. Lisaks on mitokondritel oluline roll ka näiteks kaltsiumi homöostaasis, programeeritud rakusurmas ja rasvhapete β-oksüdatsioonis. Mitokondritel on väike ja kompaktne genoom, mtDNA, millelt kodeeritakse mitokondriaalse hingamisahela komponente ning mitokondriaalseid tRNA-sid ja rRNA-sid. Inimese mtDNA on pakitud peaasjalikult TFAM valgu abil üksustesse, mida nimetatakse nukleoidideks. Nukleoidid on seotud mitokondri sisemembraaniga ning vastavalt nende funktsioonile ja kooslusele esineb vähemalt kaks nukleoidide populatsiooni. Aktiivselt replitseeruvad nukleoidid, mida iseloomustab helikaasi Twinkle esinemine, on muuhulgas seotud kolesteroolirikka membraanistruktuuriga (replikatsiooni mikrodomään). Antud struktuur on omakorda assotsieerunud ER-mitokonder ühendustega, mida nimetatakse mitokondritega assotsieerunud ER membraanideks (MAM). MAM kontaktid on mtDNA segregatsiooni seisukohast väga olulised, kuna mitokondrite jagunemine ning nukleoidide jaotumine leiab aset MAM kontaktide vahetus läheduses. Samas pole teada täpne regulatsioon, kuidas mitokondriaalsed nukleoidid mõjutavad MAM kontaktpunktide moodustumist kohas, kus toimub mtDNA süntees. Antud töö tulemused aitavad mõista nukleoidide ja organellidevaheliste kontaktide omavahelist koosmõju. Töö käigus leiti, et nukleoidi pakkimisvalgu TFAM allareguleerimisel leiab aset nukleoidide agregeerumine, oluline mtDNA koopiaarvu langus, replikatiivsete nukleoidide populatsiooni suurenemine ning MAM kontaktide ümberorganiseerimine. Antud tulemused näitavad, et nukleoidide terviklikkus on üheks oluliseks eelduseks ER-i ja mitokondri vaheliste interaktsioonide moodustmiseks. Muuhulgas heidavad käesoleva töö tulemused valgust mtDNA ning mtRNA metabolismiga seotud valkude funktsioonidele, aidates kaasa mtDNA säilitamisega seotud häirete tekkepõhjuste mõistmisele.In eukaryotic cells, mitochondria are organelles of endosymbiotic origin with a well-known function in the synthesis of ATP via oxidative phosphorylation. Mitochondria also play an important role in calcium homeostasis, programmed cell death and β-oxidation of free fatty acids. Mitochondria possess a small and compact genome, which encoded in the mtDNA, which encodes for essential subunits of the electron transport chain, mitochondrial tRNAs and rRNAs. Human mtDNA is packaged exclusively by the TFAM protein into units called nucleoids. Nucleoids are associated with the inner mitochondrial membrane and based on their composition and function at least two pools of nucleoids exist. Nucleoids actively engaged in replication and characterized by the presence of helicase Twinkle are associated with a cholesterol rich membrane structure (replication microdomain). This structure is in turn associated with ER-mitochondria junctions termed mitochondria associated ER membranes (MAM). MAM contacts are essential for mtDNA segregation, as mitochondrial division and nucleoid allocation occur in the vicinity of a subset of MAM. However, it remains to be elucidated how nucleoids impact the formation of MAM at sites of mtDNA synthesis. The results in this thesis provide further knowledge on the interplay between nucleoids and organelle tethers. It was found that a downregulation of nucleoid packaging protein TFAM results in nucleoid aggregation, significant decrease in mtDNA copy number, increased proportion of replicating nucleoids and reorganization of MAM contacts. These results show that the nucleoid integrity is prerequisite for the formation of ER-mitochondria connections. Additionally, this study helps to shed light onto the functions of the proteins involved in mtDNA and mtRNA metabolism, thereby contributing to the understanding of defects associated with mtDNA maintenance.https://www.ester.ee/record=b547832

Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity

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