A biogênese do ribossomo é um processo complexo, altamente ordenado e regulado, no qual o transcrito primário é processado por endo e exonucleases para gerar os RNAs ribossomais maduros. Este processo foi melhor caracterizado em Saccharomyces cerevisiae, porém alguns fatores atuantes em humanos tiveram uma função divergente descrita. Um desses fatores é a proteína NIP7, altamente conservada em eucariotos, que atua na formação da subunidade ribossomal 60S, em levedura, e 40S, em humanos. Assim, esse trabalho propôs a caracterização funcional da proteína CrNIP7, homóloga a NIP7, presente em Chlamydomonas reinhardtii. C. reinhardtii é uma alga verde unicelular ancestral a plantas, utilizada como modelo eucarioto para estudos de fotossíntese e de flagelos. Nesse trabalho, um estudo de complementação funcional foi realizado utilizando duas linhagens de Saccharomyces cerevisiae diferentes e em ambas CrNIP7 complementou a função de Nip7p de leveduras, indicando uma participação na síntese da subunidade 60S do ribossomo. Uma busca por parceiros de interação de CrNIP7 foi também realizada, utilizando CrNIP7 como isca para rastrear uma biblioteca de cDNA de C. reinhardtii em sistema de duplo híbrido em leveduras, o que resultou em dois novos potenciais parceiros de interação. Esses parceiros foram identificados como proteínas preditas conceitualmente no genoma de C. reinhardtii, denominadas Predicted e G-patch. Adicionalmente, a interação entre CrNIP7 e CrSBDS, proteína homóloga a Sdo1 (de levedura) e HsSBDS (de humanos), foi confirmada através de um experimento de duplo híbrido dirigido. A interação entre as proteínas CrNIP7 e CrSBDS foi validada por pull down e um teste preliminar sugeriu que CrNIP7 e Predicted também interagem in vitro. Análises de bioinformática indicam que Predicted, G-patch e CrSBDS tenham regiões intrinsicamente desordenadas, as quais podem se estruturar na interação com seus parceiros. Em conjunto, os resultados desse trabalho contribuem para entendimento do papel de CrNIP7 na biogênese de ribossomos em Chlamydomonas reinhardtii em comparação com outros modelos eucarióticos. / Ribosome biogenesis is a complex, highly regulated and ordered process in which the primary transcript is processed by endo- and exonucleases to generate the mature ribosomal RNAs. This process was best characterized in Saccharomyces cerevisiae, but some factors have been described in humans with different function. One of these divergent factors is NIP7, a highly conserved protein in eukaryotes, which acts in the formation of ribosomal 60S subunit, in yeast, and 40S, in humans. Based on this, this work proposed the functional characterization of CrNIP7 protein, homologous to NIP7, from Chlamydomonas reinhardtii. C. reinhardtii is a green alga, ancestral to plants, that is used as an eukaryote model for photosynthesis and flagella studies. In this study, a functional complementation assay was performed using two different strains of Saccharomyces cerevisiae and, in both approaches, CrNIP7 protein complemented the function of Nip7p from yeast, indicating its participation in the synthesis of the 60S ribosomal subunit. A two-hybrid assay was carried out using CrNIP7 as bait to screen a C. reinhardtii cDNA library in order to find out CrNIP7 interaction partners, wich resulted in two novel potentially partners. The interacting proteins were identified as conceptually predicted proteins in the genome of C. reinhardtii and were called Predicted and G-patch. Additionally, the interaction between CrNIP7 and CrSBDS, a protein homologous to Sdo1 (yeast) and HsSBDS (humans), was confirmed by a direct two-hybrid assay. The interaction between CrNIP7 and CrSBDS proteins was validated by pull down and a preliminary test suggested that CrNIP7 and Predicted also interact in vitro. Bioinformatics analyzes indicate that Predicted, G-patch and CrSBDS have intrinsically disordered regions, which can be ordered in the moment of interaction. Taken together, the results of this work contribute to understand the role played by CrNIP7 in ribosome biogenesis in Chlamydomonas reinhardtii compared to other eukaryotic models.

The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored. Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody against the N-terminal region of Nrap. The anti-Nrap antibody was found to detect two Nrap bands in mouse fibroblast cells, possibly corresponding to the two mouse Nrap isoforms, and . In addition, mammalian expression vectors containing the full Nrap sequence as well as deletion constructs were created. The subcellular localisation of each construct was observed by fluorescent microscopy. It was revealed that recombinant Nrap did not localise to the nucleolus, possibly because it was exported to undergo degradation by the 26S proteasome. Two putative NLSs were found to be responsible for directing Nrap to the nucleus but a region accountable for nucleolar localisation was not identified. The data indicated that multiple domains working together are likely to direct Nrap to the nucleolus. Nrap was also observed to co-localise with nucleolar proteins B23 and p19ARF. Moreover, it was shown by reciprocal immunoprecipitation that these three nucleolar proteins existed in a complex in unsynchronised mouse fibroblast cells. Recent reports demonstrated a complex relationship between B23 and p19ARF although the functional significance remained unclear. Nrap's in vivo association with B23 and p19ARF indicated a specific functional role in the nucleolus. Nrap knockdown using siRNA significantly increased B23 protein levels in a dose-dependent manner and down-regulated p19ARF protein levels at higher siRNA concentration. Preliminary studies also implicated Nrap in cell proliferation through these novel interactions. Both endogenous and recombinant Nrap were found to be highly unstable suggesting that Nrap might regulate B23 and p19ARF through its own tightly regulated stability. Finally, the role of Nrap in rRNA processing was investigated by northern blot analysis. Nrap knockdown was found to affect the levels of 45S, 32S and 28S rRNAs. The changes found may be a consequence of the concurrent perturbation in the levels of B23 and p19ARF caused by Nrap knockdown. As the results were not consistent with previous reports, it was likely that changes to rRNA processing could be contributed to Nrap loss of function. This study demonstrated for the first time a functional role of Nrap in rRNA processing possibly through its association with B23 and p19ARF.

Approximately 10% of the open reading frames in the genome of the Gram-negative bacterium E. coli encodes secretory proteins, and 20% encodes integral inner membrane proteins (IMPs). These proteins are sorted to their correct cellular compartments (the periplasm and the outer and inner membranes) by specialized targeting and translocation/insertion systems. So far, a very limited set of model proteins have been used to study proteins sorting requirements in E. coli. The main objective of all the papers presented in this thesis was to determine the targeting and translocation/insertion requirements of more E. coli proteins. In papers I and II, this was done using focused approaches. Selected model proteins (lipoproteins and putative outer membrane proteins) were expressed from plasmids and their targeting and translocation were analysed in vitro by crosslinking experiments and/or in vivo by pulse-chase analysis in different E. coli mutant strains. In papers III a comparative sub-proteome analysis was carried out to define the role of the cytoplasmic chaperone SecB in protein targeting. In paper IV, a similar approach was used to study how protein translocation and insertion is affected upon depletion of the essential Sec-translocon component SecE. The ‘global’ approach used in paper III and IV allowed us to study protein targeting and translocation/insertion requirements on a proteome level. This led to the identification of several novel SecB substrates and a large number of potential Sec-translocon independent IMPs.

The DrrAB proteins of Streptomyces peucetius belong to the ABC family of ubiquitous membrane transporters. The DrrA and DrrB proteins together form a drug efflux pump that carries out the transport of the anticancer drug doxorubicin by carrying out ATP hydrolysis. The present study is the first where the intrinsic factors involved in the assembly of the DrrAB functional complex have been elucidated. The drrA and drrB genes in the wild type operon have overlapping stop and start codons (ATGA) which indicates translational coupling between the two genes. On insertion of a fortuitous stop codon in DrrA it was shown that the expression of DrrB is coupled to that of the upstream gene drrA. Furthermore, it was observed that a functional complex could be achieved only when the genes were maintained in cis in a translationally coupled manner. Translational regulation in DrrA was found to be involved in the control of optimal levels of DrrB. Inhibitory interactions within drrA sequence were speculated to cause translational arrest at the C terminus of DrrA. A novel assembly domain that forms the interface between DrrA containing the Nucleotide Binding Domain (NBD) and DrrB comprising the TransMembrane Domain (TMD) was found. Based on the data presented in this study a model is proposed for the biogenesis of the DrrAB drug pump. The model suggests that translational coupling between DrrA and DrrB is crucial for functional complex formation. Further, there is evidence of regulation of translation by attenuation in the intergenic region of drrA and drrB. The regulation seems to involve the last 30 nucleotides of the mRNA of drrA and some upstream sequences within drrA that cause translational arrest within the C terminus of DrrA. Since DrrB is translationally coupled to drrA, this translational arrest in conjunction with coupling causes lowering in the levels of DrrB. Finally, since the DrrA-DrrB interaction domain lies in the C terminus of DrrA, only the fully translated DrrA product will be competent to form a complex with DrrB. This interaction between the C terminus of DrrA and the N terminus of DrrB may be crucial for initial targeting of the complex to the membrane. The model is expected to serve as primer and open up an interesting yet insufficiently understood subject of membrane protein biogenesis.

Mitochondria play an essential role in cell metabolism as well as apoptotic signaling. Chronic endurance exercise has been shown to increase mitochondrial content in skeletal muscle. Interestingly, endurance exercise has also been associated with decreased skeletal muscle apoptosis; however, the direct effect of increased skeletal muscle mitochondrial content on apoptotic signaling has not been examined. The purpose of this study was to induce mitochondrial biogenesis in L6 myoblasts and examine the susceptibility of these cells to stress- induced apoptosis. Mitochondrial biogenesis was accomplished using 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and S-nitroso-N-acetylpenicillamine (SNAP), which activate AMPK and donate nitric oxide, respectively. Successful induction of mitochondrial biogenesis was determined by western blot analysis for mitochondrial specific markers. Following SNAP and AICAR treatment, the average increase in the mitochondrial markers was 24% and 38%, respectively. Subsequent exposure of cells to several apoptosis-inducing agents increased apoptosis. Interestingly, SNAP- and AICAR- treated cells had a lower percentage of apoptotic cells as determined by AnnexinV-FITC/PI fluorescent staining, cell cycle analysis, and cell counting/size analysis. In addition, it was shown that SNAP- and AICAR-treated cells had reduced caspase-3 activity following exposure to apoptotic stimuli. Furthermore, treatment with SNAP and AICAR resulted in increased protein content of the antioxidants MnSOD and catalase. Interestingly, mitochondrial ROS production was not significantly altered between groups with total cellular ROS production being increased in the SNAP- and AICAR-treated groups. In summary, this work demonstrates that increasing mitochondrial content in L6 myoblasts provides protection against stress-induced apoptosis. The mechanism for this protective effect remains to be determined; however, it may be mediated by a combination of increased antioxidant capacity and improved mitochondrial calcium buffering capacity.

The purpose of this study was to investigate the effects of hydroxytyrosol (HT) supplementation on markers of mitochondrial biogenesis, aerobic capacity, and endurance exercise performance in recreationally active men. Sixty-one (n = 61) subjects (21.46 ± 0.22 yrs, 179.46 ± 0.79 cm, 78.91 ± 1.19 kg) consumed either a high dose (HI) HT supplement (150 mg HT), a low dose (LO) HT supplement (50 mg HT), or a placebo (PLA) every day for 6 weeks. Muscle biopsies from the vastus lateralis were obtained at baseline and after 6 weeks of supplement consumption and analyzed for markers of mitochondrial biogenesis: succinate dehydrogenase (SDH), citrate synthase (CS), and peroxisome proliferator-activated receptor ɣ coactivator (PGC)-1α. Subjects completed exercise testing on a bicycle ergometer at baseline and after 3 and 6 weeks of supplement consumption to measure changes in maximal aerobic power (VO2MAX), lactate threshold, respiratory exchange ratio (RER), substrate utilization, and endurance exercise performance on a 20 km time trial course. The primary findings were that HT supplementation increased muscle oxidative enzyme activity suggesting increased oxidative capacity. HT also increased time trial performance at midpoint and endpoint and this corresponded with an improvement in lactate threshold and a lower RER for the LO HT treatment. Time trial performance was also improved at endpoint for PLA, however, unlike LO an HI HT, this was accompanied by a significant increase in rating of perceived exercise (RPE) and not associated with improvements in muscle oxidative capacity. Our results indicate that HT ranging from 50 to 150 mg/day for 6 weeks can improve muscle oxidative capacity and aerobic performance, and suggests that HT may be used chronically to improve mitochondrial function. HT may be used as an effective means to increase mitochondria to improve exercise performance, and limit diseases associated with mitochondrial dysfunction such as cardiovascular disease, type II diabetes, and some cancers. / text

The RNA helicase Prp43p is one of the few members of the DEAH-box helicase family that is known to operate in more than one cellular process in Saccharomyces cerevisiae. With roles in ribosome biogenesis and pre-mRNA splicing, Prp43p may be important in maintaining a communication conduit between these two pathways. Our studies provide insights into how Prp43p function is regulated through the use of three cofactors, Ntr1p, Pfa1p, and Gno1p, all of which interact with Prp43p at different steps of pre-mRNA splicing or ribosome biogenesis. Each cofactor contains a unique G-patch domain and our data show that they associate with Prp43p in a mutually exclusive manner. A strong growth defect and RNA processing phenotypes are seen upon overexpression of Pfa1p due to the dominance of Pfa1p interaction with Prp43p. Moreover, excess Pfa1p precludes Prp43p from interacting with either 35S pre-rRNA or U6 snRNA, indicating this one cofactor can negatively regulate Prp43p recruitment into ribosome biogenesis and pre-mRNA splicing pathways, respectively. We have determined that Ntr1p and Gno1p are able to compete with one another for Prp43p occupancy. Similar to Ntr1p, we show that the G-patch domain of Gno1p contributes to its association with Prp43p. To further understand pathway specificity of Prp43p, we characterized conditional prp43 alleles with mutations C-terminal to the conserved RecA domains of Prp43p. These novel alleles affect pre-mRNA splicing and ribosome biogenesis, though none are mutually exclusive. Multiple prp43 alleles are deficient in tri-snRNP formation, a previously uncharacterized phenotype in prp43 mutants. The majority of our prp43 mutants display varying rRNA defects, with some alleles impacting ribosome biogenesis more severely or moderately than known prp43 ATPase mutants. To correlate the processing defects seen in each allele, we have determined the extent of association of the mutants with each G-patch protein. Altogether, our data support a working model for Prp43p in which its substrate specificity, activation, and cellular distribution is coordinated through the efforts of the three G-patch proteins in yeast and sheds light on potential mechanisms of general DExH/D helicase function and regulation. / text

Skeletal muscle possesses remarkable plasticity in responses to altered mechanical load. An established murine model used to increase mechanical load on a muscle is the surgical removal of the gastrocnemius and soleus muscles, thereby placing a functional overload on the plantaris muscle. As a consequence, there is hypertrophic growth of the plantaris muscle. We used this model to study the molecular mechanisms regulating skeletal muscle hypertrophy. Aged skeletal muscle demonstrates blunted hypertrophic growth in response to functional overload. We hypothesized that an alteration in gene expression would contribute to the blunted hypertrophic response observed with aging. However, the difference in gene expression was modest, with cluster analysis showing a similar pattern of expression between the two groups. Despite ribosomal protein gene expression being higher in the aged group, ribosome biogenesis was significantly lower in aged compared with young skeletal muscle in response to the hypertrophic stimulus (50% versus 2.5-fold, respectively). The failure to fully up-regulate pre-47S ribosomal RNA (rRNA) expression in old skeletal muscle undergoing hypertrophy indicated ribosomal DNA transcription by RNA polymerase I was impaired. Contrary to our hypothesis, the findings of the study suggest that impaired ribosome biogenesis was a primary factor underlying the blunted hypertrophic response observed in old skeletal muscle rather than dramatic differences in gene expression. As it appears ribosomal biogenesis may limit muscle hypertrophy, we assessed the dynamic changes in global transcriptional output during muscle hypertrophy, as the majority of global transcription is dedicated to ribosome biogenesis during periods of rapid growth. Metabolic labeling of nascent RNA using 5-ethynyl uridine permitted the assessment of cell type specific changes in global transcription and how this transcription is distributed within the myofiber. Using this approach, we demonstrate that myofibers are the most transcriptionally active cell-type in skeletal muscle, and furthermore, myonuclei are able to dramatically upregulate global transcription during muscle hypertrophy. Interestingly, the myonuclear accretion that occurs with hypertrophy actually results in lower transcriptional output across nuclei within the muscle fiber relative to sham conditions. These findings argue against the notion that nuclear accretion in skeletal muscle is necessary to increase the transcriptional capacity of the cell in order to support a growth response.

Mitochondria play an essential role in cell metabolism as well as apoptotic signaling. Chronic endurance exercise has been shown to increase mitochondrial content in skeletal muscle. Interestingly, endurance exercise has also been associated with decreased skeletal muscle apoptosis; however, the direct effect of increased skeletal muscle mitochondrial content on apoptotic signaling has not been examined. The purpose of this study was to induce mitochondrial biogenesis in L6 myoblasts and examine the susceptibility of these cells to stress- induced apoptosis. Mitochondrial biogenesis was accomplished using 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and S-nitroso-N-acetylpenicillamine (SNAP), which activate AMPK and donate nitric oxide, respectively. Successful induction of mitochondrial biogenesis was determined by western blot analysis for mitochondrial specific markers. Following SNAP and AICAR treatment, the average increase in the mitochondrial markers was 24% and 38%, respectively. Subsequent exposure of cells to several apoptosis-inducing agents increased apoptosis. Interestingly, SNAP- and AICAR- treated cells had a lower percentage of apoptotic cells as determined by AnnexinV-FITC/PI fluorescent staining, cell cycle analysis, and cell counting/size analysis. In addition, it was shown that SNAP- and AICAR-treated cells had reduced caspase-3 activity following exposure to apoptotic stimuli. Furthermore, treatment with SNAP and AICAR resulted in increased protein content of the antioxidants MnSOD and catalase. Interestingly, mitochondrial ROS production was not significantly altered between groups with total cellular ROS production being increased in the SNAP- and AICAR-treated groups. In summary, this work demonstrates that increasing mitochondrial content in L6 myoblasts provides protection against stress-induced apoptosis. The mechanism for this protective effect remains to be determined; however, it may be mediated by a combination of increased antioxidant capacity and improved mitochondrial calcium buffering capacity.

No description available.