Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 99-127).

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains ix, 103 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 89-100).

Curcumin is one of the major curcuminoids produced by the ginger family Zingiberaceae. These curcuminoids possess pharmacological properties that include anticancer activities. We have evaluated some synthetic curcumin analogues that have shown potential as anticancer drugs. These antineoplastic agents bearing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore are electrophiles which are designed to preferentially react with sulfhydryl groups present in proteins as opposed to amino and hydroxyl groups present in DNA. In previous pilot studies, three derivatives examined in this thesis showed inhibition towards human cancer cell lines such as Molt 4/C8 and CEM T-lymphocytes. In this thesis work, I determined the cytotoxicity of these derivatives and curcumin towards human colon cancer (HCT-116) cells and also normal colon epithelial (CRL-1790) cells, and examined the possible mechanism(s) involved. I hypothesized that they act via induction of reactive oxygen species (ROS) which elicit a transient surge of mitochondrial ROS generation and a phenomenon known as ROS-induced ROS release (RIRR), along with the mitochondrial permeability transition (MPT) and mitochondrion –dependent apoptosis. I asked whether these agents react with some of the key protein thiols in the mitochondria whose oxidation/alkylation results in mitochondrion - dependent apoptosis. NC-2109 and NC-2346 were found to be potent cytotoxic agents based on their GI50 values of 0.87 ± 0.38 μM and 0.90 ± 0.22 μM, respectively, and were more potent than the anticancer drug 5-fluorouracil (GI50 = 5.47 ± 0.55 μM) and curcumin (GI50 = 3.50 ± 0.36 μM). However NC-2109 was found to have a better selectivity towards cancer cells over normal cells (a selectivity index of 18.81 versus 5-FU, curcumin and NC-2346 which had selectivity indices of 1.87, 16.75 and 4.61, respectively). In the investigations of the mechanisms involved, both curcumin and curcumin analogues were able to induce mitochondrial ROS production. Moreover, curcumin and its synthetic counterparts showed a biphasic ROS profile which is most characteristic of RIRR. Treatment with these agents also led to the disruption of the mitochondrial membrane potential, suggesting oxidation of protein thiols and the opening of the mitochondrial permeability transition pore which is an important step to initiate mitochondria-directed apoptosis. This possibility was confirmed based on GSSG/GSH ratios, since curcumin, NC-2346 and NC-2109 all produced a higher GSSG/GSH ratio than the controls. In addition to their ability to depolarize the mitochondrial membrane in HCT-116 cells, that these molecules acted via the mitochondrial pathway were further authenticated based on their ability to induce mitochondrial swelling in rat liver mitochondria. In another part of this thesis I evaluated the involvement of the critical thiol protein adenine nucleotide translocase (ANT), a bifunctional protein that plays a central role in mitochondrial apoptosis. ANT has four different isoforms; ANT1 and ANT3 are proapoptotic, while ANT2 and ANT4 are antiapoptotic and are overexpressed in cancer states. A combination approach using ANT2 siRNA however did not conclusively show whether these agents acted synergistically with ANT2 knockdown to potentiate mitochondria-mediated cell death. An alternative combination approach was the use of carboxyatractyloside (CAT) which binds to and retains ANT in its ‘c’ conformation, exposing thiols and potentially driving a cell towards programmed cell death. The presence of CAT enhanced the ability of curcumin and its synthetic analogs to collapse the mitochondrial membrane potential, an important step in mitochondrial-mediated apoptosis. In conclusion, curcumin and the curcumin analogue NC-2109 were found to be cytotoxic in vitro, towards HCT-116 cells and also showed good selectivity. In addition, these two molecules were found to be ROS inducers, and coincidentally oxidized cellular thiols and caused depolarization of the mitochondrial membrane potential. The results support a mechanism of mitochondrial-mediated cell death upon MPT pore formation (mitochondrial swelling), perhaps involving ANT2. This conclusion was further supported by the potentiation of cell death in the presence of the ANT2 inhibitor, CAT.

No description available.

<p>Zahvaljujući amfifilnoj strukturi i mogućnosti građenja konjugata, žučne kiseline - endogeno sintetisani produkti katabolizma holesterola su prepoznate kao potencijalni nosači lekova i promoteri transporta kroz biolo&scaron;ke membrane. Otkriće da aktivacijom specifičnih nuklearnih receptora reguli&scaron;u ekspresiju gena uključenih u plejadu signalnih puteva uključenih u metabolizam, proliferaciju i diferencijaciju ćelija i onkogenezu, pro&scaron;irilo je ulogu žučnih kiselina u odnosu na inicijalno opisanu ulogu intestinalnih emulgatora. Žučne kiseline se danas ne smatraju samo pasivnim nosačima lekova i promoterima transporta kroz biolo&scaron;ke membrane već i molekulima sa farmakodinamskom funkcijom, koji reguli&scaron;u različite aspekte integrativnog ćelijskog metabolizma. Doksorubicin je jedan od najče&scaron;će kori&scaron;ćenih antineoplastičkih agenasa i sastavna je komponenta mnogih hemoterapijskih protokola u lečenju solidnih i hematolo&scaron;kih maligniteta. Međutim, hepatotoksični i kardiotoksični efekti značajno ograničavaju upotrebu ovog, inače veoma korisnog antitumorskog agensa. Pojava odložene dozno-zavisne kardiotoksičnosti predstavlja značajan zdravstveni problem onkolo&scaron;kih pacijenata sa uspe&scaron;no lečenim malignitetom, naročito pacijenata lečenih u pedijatrijskom uzrastu. Budući da je razvoj novih lekova veoma dug i skup proces sa neizvesnim ishodom, pobolj&scaron;anje farmakodinamskih i farmakokinetskih svojstava već postojećih antitumorskih agenasa sa dokazanom efikasno&scaron;ću, uz smanjenje toksičnih efekata, predstavlja racionalan istraživački pristup u savremenoj medicini. Osnovni cilj ovog rada je ispitivanje uticaja žučnih kiselina ursodeoksiholne, henodeoksiholne i 12-okso-henodeoksiholne kiseline (12-monoketoholne kiseline) na citotoksičnu aktivnost doksorubicina prema MCF-7 ćelijskoj liniji humanog adenokarcinoma dojke i ispitivanje molekularnih mehanizama odgovornih za farmakodinamske efekte. Takođe su navedene žučne kiseline ispitane kao promoteri transporta koji utiči na prodor i kumulaciju doksorubicina u malignim ćelijama. U ovom radu je ispitan uticaj koadministracije navedenih žučnih kiselina sa doksorubicinom na odložene toksodinamske efekte (hepatotoksičnost i kardiotoksičnost) kod pacova, ali i efekti pretretmana žučnim kiselinama na koncentracije doksorubicina u krvi, bilijarnu ekskreciju leka kao i kumulaciju u jetri i miokardu eksperimentalnih životinja. Žučne kiseline su u netoksičnim koncentracijama potencirale in vitro citotoksične efekte doksorubicina na MCF-7 ćelijskoj liniji pri čemu je henodeoksiholna ispoljila sinergistički efekt, dok su ursodeoksiholna u 12-monoketoholna ispoljile aditivni citotoksični efekt sa doksorubicinom. Ispitivanjem molekularnih mehanizama citotoksičnih efekata utvrđeno je da su žučne kiseline u različitom stepenu potencirale apoptozu ćelija mitohondrijalnim putem uticajem na ekspresiju pro- i antiapoptotskih proteina na transkripcionom nivou i povećale stres endoplazmatskog retikuluma, ali i dovele do alteracija ekspresije gena koji kodiraju sintezu antioksidativnih enzima, transmembranskih efluks proteina i enzima uključenih u metaboličku inaktivaciju leka. Žučne kiseline u netoksičnim koncentracijama su takođe značajno povećale prodor i kumulaciju doksorubicina u MCF-7 ćelijskoj liniji. U in vivo sistemu, koadministracija žučnih kiselina nije rezultovala u pobolj&scaron;anju odloženih toksodinamskih efekata visokih doza doksorubicina na biohemijskom i molekularnom nivou. Međutim, nakon pretretmana žučnim kiselinama, vrednosti koncetracija doksorubicina u serumu su bile povi&scaron;ene nakon pretretmana urso- i henodeoksiholnom kiselinom i snižene nakon pretretmana 12-monoketoholnom kiselinom uz povećanje bilijarne sekrecije doksorubicina. Pored promena u farmakokinetskom profilu doksorubicina, pretretman žučnim kiselinama je blago redukovao prodor i kumulaciju doksorubicina u hepatocite i kardiomiocite. Na osnovu rezultata ove studije može se zaključiti da primena ispitivanih žučnih kiselina sa doksorubicinom povećava prodor i pobolj&scaron;ava farmakodinamski profil doksorubicina in vitro, na ćelijskom modelu humanog adenokarcinoma dojke. Pobolj&scaron;anje selektivnog preuzimanja i prodora doksorubicina u maligne ćelije koje nije praćeno povećanom kumulacijom u normalnim tkivima, kao i pobolj&scaron;anje antitumorskog dejstva doksorubicina sa mogućim smanjenjem doze uz smanjenje pojave dozno-zavisnih neželjenih dejstava doksorubicina čini žučne kiseline molekulima kandidatima za dalja ispitivanja u cilju razvoja novih, pobolj&scaron;anih antitumorskih terapijskih strategija.</p> / <p>Due to the amphiphilic structure and the significant conjugation potential, bile acids - endogenously synthesized products of cholesterol catabolism have been recognized as potential drug carriers and promoters of transport through biological membranes. The discovery that by activating specific nuclear receptors bile acids regulate the expression of genes involved in various signaling pathways including metabolism, cell proliferation and differentiation as well as carcinogenesis, expanded initially ascribed role of intestinal emulsifiers to the various fields. Bile acids are now considered not to act only as passive carriers of drugs and promoters of transport through biological membranes, but also as the molecules with pharmacodynamic activity, regulating various aspects of integrative cellular metabolism. Doxorubicin is one of the most commonly prescribed antineoplastic agents as an integral component of many chemotherapy protocols in the treatment of both solid and hematologic malignancies. However, hepatotoxic and cardiotoxic effects significantly limit the use of this, otherwise, very useful anti-tumor agent. The development of dose-dependent cardiotoxic side effects represents particular health issue in successfully treated oncological patients, especially among survivors of pediatric malignancies. Since the development of new drugs is very long and expensive process with an uncertain outcome, improving the pharmacodynamic and pharmacokinetic properties of the existing agents with proven efficacy, while reducing toxic side effects, represents a rational approach to research in modern medicine. The main objective of this work is to examine the role of bile acids: ursodeoxycholic, chenodeoxycholic and 12-oxo-chenodeoxycholic acid (12-monoketocholic acid) on the cytotoxic activity of doxorubicin in the MCF-7 human breast adenocarcinoma cell line, and to get insight on molecular mechanisms responsible for underlying pharmacodynamic effects. The capacity of bile acids to promote the transport and accumulation of doxorubicin in malignant cells was also evaluated. In addition, the effect of co-administration of the bile acids with doxorubicin on delayed toxodynamic effects (hepatotoxicity and cardiotoxicity) in rats, as well as the effects of bile acid pretreatment on the doxorubicin serum concentration and pharmacokinetic profile, biliary excretion of the drug as well as accumulation in the liver and myocardial cells of experimental animals were examined. Bile acids applied in non-toxic concentrations potentiated in vitro cytotoxic effects of doxorubicin in MCF-7 cell line. Chenodeoxycholic acid exhibited a synergistic effect, whereas ursodeoxycholic and 12-monoketocholic acid exhibited an additive cytotoxic effect with doxorubicin. By examining the underlying molecular mechanisms of cytotoxic effects, bile acids have been found to potentiate apoptosis of cells by mitochondrial-dependent pathway by modifying the expression of pro- and anti-apoptotic proteins at the transcriptional level and to increase endoplasmic reticulum stress, but also have altered the expression of genes encoding the synthesis of antioxidant enzymes, transmembrane efflux proteins and enzymes involved in metabolic inactivation of the drug. Non-toxic concentrations of bile acids also significantly increased the penetration and accumulation of doxorubicin in MCF-7 cell line. In the in vivo system, the co-administration of bile acid did not improved delayed toxodynamic effects of high dose of doxorubicin both at the biochemical and molecular levels. However, pretreatment with bile acids resulted in alterations of serum doxorubicin concentrations. Chenodeoxycholic and ursodeoxycholic acid elevated whereas 12-monoketocholic acid decreased serum doxorubicin concentrations. In addition to changing pharmacokinetic profile of doxorubicin on bile acid species-dependent manner, all bile acids have also increased excretion of drug by the biliary route, and slightly reduced penetration and accumulation of doxorubicin in hepatocytes and cardiomyocytes. Based on the results of this study, the administration of the examined bile acids with doxorubicin increases the penetration and improves the pharmacodynamic profile of doxorubicin in vitro on the cell model of human breast adenocarcinoma. The improvement of selective uptake and penetration of doxorubicin into malignant cells that is not accompanied by increased accumulation in normal tissues, as well as the improvement in the anti-tumor effects of doxorubicin with a possibility to reduce the dose and thereby the occurrence of dose-dependent undesirable effects of doxorubicin, render bile acids as the potential candidate molecules in developing novel antitumor therapeutic strategies.</p>

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 112-125). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

The present thesis focuses on the elucidation of human mitochondrial inner membrane presequence-translocation machinery with implications on cancer cell proliferation. Mitochondria are the endosymbiotic organelles in an eukaryotic cell performing a vast repertoire of functions and require approximately 1500 proteins. However, the mitochondria genome contains only 13 protein-coding genes primarily transcribing the complexes of the electron transport chain. Therefore, it is evident that most of the mitochondrial proteome is encoded by the nucleus and synthesized on cytosolic ribosomes. Chapter 1: Mechanism of mitochondrial inner membrane protein translocation and its oncogenic connection. Mitochondria consist of different routes of directing proteins to their intramitochondrial destinations. The presequence pathway, mediated by the inner membrane TIM23 complex, is responsible for the import of matrix and a number of single transmembrane helixes containing inner membrane proteins. This pathway accounts for approximately 60% of the total proteome imported into the organelle and hence, is the major focus of discussion in the present study. The components of the TIM23 complex can be subdivided into two groups, the protein conducting channel and the import motor. The initial translocation across the TIM23 channel utilizes the electrochemical membrane potential that exists across the inner membrane whereas the final step of the translocation process is driven by energy from ATP hydrolysis. MtHsp70 forms the central component of the import motor, and its function is regulated by the J-proteins. Pam18 stimulates the ATPase activity of mtHsp70. Pam16, on the other hand, forms a subcomplex with Pam18 and exerts an inhibitory effect its ATPase stimulatory activity, in turn regulating the activity of the import motor. The stoichiometric coupling with the substrate binding-release cycle of mtHsp70 drives the import process. Although the organization of presequence translocation machinery and its functional annotations have been described in detail in yeast system, little information is available on its organization in human. It is difficult to contemplate the existence of similar machinery in human mitochondria with complex and diversified functions. Human mitochondria apart from regulating the metabolic pathways are involved in progression of cancer, neurodegenerative disorders, responses to xenobiotic stress and induction of apoptosis. Numerous reports have shown that mutations and overexpression of human orthologs of translocase components are associated with various cancer subtypes. Such disease condition also involves targeting of specific cell signaling molecules that reprogram organellar functions and alter the cellular phenotype. Based on this evidence we defined our study into four broad objectives – 1) identify the components of human presequence translocase as Chapter two and three, 2) characterize the subunit organization of human presequence translocation machinery in Chapter four, 3) determine the functional connection between the translocase components and the cancer phenotype in Chapter four and five and 4) understand how the functions of J-proteins have evolved across the species as Chapter six. Chapter 2: Unraveling the role of Magmas in human mitochondrial protein transport. Pam16 plays a critical role in regulation of import process by governing the activity of the import motor. Proteins orthologous to Pam16 had been reported earlier to be overexpressed in various metabolically active tissues and cancer subtypes. We found that in humans a protein named as Mitochondria Associated Granulocyte Macrophage colony Stimulating factor signaling molecule (Magmas) showed significant sequence similarity with yeast Pam16 at its C-terminal region. Magmas was initially discovered as a protein that was overexpressed in neoplastic prostrate and when the cells were exposed to GM-CSF. Our experiments suggested that Magmas localized in human and yeast mitochondria and it was associated with the inner mitochondrial membrane. Magmas could complement the growth of yeast cells that were deleted for the essential gene PAM16 and could import precursor proteins into the mitochondria. Like Pam16, Magmas was able to form a stable heterodimeric subcomplex with yeast Pam18 and human Pam18 ortholog DnaJC19 (JC19). We found that J-domain forms the minimal region required for heterodimer formation between Magmas and Pam18/JC19. Mutations in Magmas J-like domain resulted in temperature sensitive growth phenotypes in yeast cells and associated import defect in translocating precursor proteins into the organelle due to inability to form a stable subcomplex with Pam18 and JC19, resulting in loss of import function. Loss of subcomplex formation leads to dissociation of Pam18 from the translocation machinery highlighting the importance of Magmas in tethering Pam18/JC19 to the presequence translocase. Magmas, showing characteristic of a J-like protein, was unable to stimulate the ATPase activity of mtHsp70. However, it exerted an inhibitory effect on the ATP stimulatory effect of the J-protein Pam18/JC19, indicating that Magmas has a regulatory effect on the overall activity of import motor. In contrast Magmas mutants those are incapable of forming a stable heterodimer with Pam18 were unable to regulate the activity of Pam18 resulting in import defects. In summary, our results highlight that Magmas is an ortholog of yeast Pam16 performing similar functions at the import channel. Chapter 3: Existence of two J-protein subcomplexes at the translocation channel with distinct physiological functions. JC19 has been regarded as the human ortholog of Pam18 whose loss of function was associated with dilated cardiomyopathy and ataxia syndrome. However, immunoprecipitation analysis using anti-Magmas antibody revealed the presence of a second J-protein identified as DnaJC15 (JC15) that shared a highly similar J-domain with JC19. JC15 was initially identified as a protein whose loss in expression resulted in development of a chemoresistant phenotype in ovarian carcinoma cells exposed to chemotherapeutic treatment. We found that JC15 localizes in mitochondria where it was associated with the inner membrane. Similar to Pam18 and JC19, JC15 heterodimerized with Magmas/Pam16 through its J-domain and associated with the presequence translocase of the inner membrane. A loss of function mutation at the J-domain of JC15 destabilizes its interaction with Magmas resulting in protein translocation defects and temperature-sensitive growth phenotype in yeast cells. The JC15 mutant showed inability to get associated with the translocation channel and had dysregulated stimulation of mtHsp70 activity leading to decreased mitochondria biogenesis and loss of mitochondrial membrane potential. In summary, our results showed that JC15 is the second human ortholog of Pam18 with similar functions. In contrast to yeast, in human mitochondria JC15 and JC19 were found to form two separate and distinct J-protein subcomplexes with Magmas at the mitochondrial import motor. The essentiality of the J-proteins for normal human mitochondria function was addressed through siRNA mediated downregulation of Magmas, JC19 and JC15. We found that Magmas and JC19 are essential for normal mitochondrial function and cell viability whereas JC15 is dispensable and might have a supportive role. Interestingly, both JC19 and JC15 interacted with Magmas with equal affinity and stimulated mtHsp70’s ATPase activity by equivalent levels. This shows that both JC19 and JC15 share similar properties in terms of their functions at the import channel, and the differences might be in a much broader perspective in terms of their association with the translocation channel. Chapter 4: Architecture of human mitochondrial inner membrane presequence -translocation machinery. In yeast, there exists a single J-protein subcomplex formed by Pam16 and Pam18, which is recruited to the sole translocase. However, humans present a completely different scenario where there exists a two distinct subcomplexes formed by Magmas with either of the J-proteins. So the question arises how the individual subcomplexes is recruited to the translocation machinery; whether they are associated to one or differentially recruited to two different translocases. We identified the existence of three distinct translocases in the human system constituted by the two J-proteins along with the Tim17 paralogs. JC15 along with Tim17a forms the translocase A of size similar to that of the yeast system, and it forms the ancestral translocase in the humans. Tim17b isoforms, on the other hand, associates with JC19 to form mammalian specific translocases B1 and B2. The association of the J-proteins at the translocation channel was found to be mediated by Magmas as a subcomplex. Downregulation of Magmas resulted in dissociation of both the J-proteins, and its overexpression resulted in redistribution of J-proteins at the translocases. We found that translocase B imported precursor proteins at a comparatively higher rate as compared to translocase A. Disruption of translocase B had deleterious effects on cell viability, respiratory chain complex's activities, Fe-S cluster biogenesis, mitochondria morphology, regulation of free radical levels and maintenance of mitochondrial genome. In contrast, depletion of translocase A did not significantly alter the survivability of cells, mitochondrial activity and maintenance of organellar morphology. This shows that translocase B is essential and performs the constitutive import function in the mammalian system whereas translocase A is dispensable and might have a supportive role in maintenance of mitochondrial function. However, translocase A play a specific role in human mitochondria in context to cancer cells. We observed that the elevated level of Tim17a found in cancer cells is responsible for maintenance of higher mitochondrial DNA copy number and higher proliferative potential of cancer cells. Additionally, translocase A also plays a specific role in translocation of cell signaling proteins that lack a mitochondrial targeting sequence into the mitochondria, highlighting the possible role of this translocase in neoplastic transformation. Chapter 5: Mechanistic insights into the role of JC15 as a part of translocase A in chemoresistant phenotype. JC15 had been initially identified to be associated with development of chemoresistance in cancer cells. However, the molecular mechanism followed by the protein has not been elucidated yet. Our studies have shown that overexpression of JC15 leads to increased sensitivity of cells to chemotherapeutic drug cisplatin and are coupled with complete loss of membrane potential, mitochondrial swelling and cytochrome c release. However, this chemosensitive phenotype was partially ameliorated upon preexposing the cell to cyclosporine A which is an inhibitor of cyclophilin D, a critical component of mitochondrial membrane transition pore (MPTP) complex. A similar reversal of phenotype was observed upon depleting cyclophilin D even under JC15 overexpressing background. This highlighted a possible functional connection between these two proteins. In order to check this hypothesis other way around, we overexpressed cyclophilin D in the cells which resulted in constitutive opening of the MPTP complex, enhanced mitochondrial swelling and reduced cell viability. In contrast, the gain of function anomalies of cyclophilin D overexpression was significantly reversed upon JC15 depletion. We observed through co-immunoprecipitation analysis that JC15 activates cyclophilin D by releasing it from the inhibitory effects of TRAP1 and couples it to the MPTP complex. Additionally, we have also shown that the J-domain of JC15 is critical for its interaction with cyclophilin D and loss of function mutation at the J-domain of JC15 disrupts its interaction with cyclophilin D. As a result the JC15 mutant is not able to mount a chemosensitive response to cisplatin drug. Chapter 6: Identification of regions determining the divergence of J-proteins functions at the mitochondrial import motor. The above studies show ample evidence to suggest that the two human J-proteins have undergone significant divergence in their function in human mitochondria in spite of having a highly similar J-domain. Therefore, we asked the question that how the human J-proteins have evolved and diversified from the primitive yeast protein Pam18 and what are the regional determinants in the protein sequence that dictate the function of the J-domain. We utilized a purely genetic approach to address the problem. We observed that JC19 was unable to rescue the growth of yeast cells deleted for the essential gene Pam18 and JC15 expression resulted in cold sensitive phenotype. We used JC15 as the model protein for our assays and applied three methodologies. First, generation and isolation of a series of mutations in JC15 that could rescue the cold sensitive phenotype, and the growth of the cells were similar to the wild type. Second, to identify the regulatory residues by isolation of second site suppressors that could be the suppressor the mutant phenotypes isolated earlier. Third, we utilized a purely evolutionary approach by swapping the individual domains between the three J-proteins- Pam18, JC19 and JC15. Our genetic data support the idea that the partial loss of function of human J-protein in the yeast system is due to altered subcomplex dynamics with Pam16. The altered dynamics of the subcomplex is mainly regulated by the residues in the arm, linker and helical regions of the J-domain, especially the helix II regions. Our analysis has also uncovered a critical role of the targeting (T) region of J-proteins which along with inter-membrane space (IMS) domain share significant sequence diversity among J-proteins in yeast and humans. The T-region in conjunction with the IMS domain plays a crucial role in regulating the J-domain’s function across the kingdoms and within the species. Although, our genetic data needs to be supplemented with biochemical evidence, this study provides significant insights into the diversity of J-protein function across the species and mode of their regulation through regions flanking the J-domain.

1 Abstract in English Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. However, why modern ETC-targeted compounds are tolerated on the organismal level and what are the molecular reasons for this tolerance remains unclear. Most somatic cells are in a non-proliferative state, and features associated with the ETC in quiescence might therefore contribute to specificity. Thus, we investigated the ETC status and the role of two major consequences of ETC blockade, reactive oxygen species (ROS) generation and inhibition of ATP production, in cell death induction in breast cancer cells and in proliferating and quiescent non-transformed cells. First, we characterised the effect of a newly developed ETC inhibitor mitochondria- targeted tamoxifen (MitoTam) in in vitro and in vivo tumour models of breast cancer with varying status of the Her2 oncogene. We document that Her2high cells and tumours have increased assembly of respiratory supercomplexes (SCs) and increased complex I-driven respiration in vitro and in vivo. They are also highly sensitive to MitoTam. Unlike the parental compound tamoxifen, MitoTam efficiently suppressed experimental Her2high tumours without systemic toxicity. Mechanistically, MitoTam inhibits complex I- driven respiration and disrupts respiratory...