Amyloidoses comprise a group of gain-of-toxic function protein misfolding diseases, in which normally soluble proteins in their functional state undergo conformational changes into highly organized and generally intractable thread-like aggregates, termed amyloid fibrils. These structures accumulate predominantly in the extracellular space but growing evidence suggests that amyloids may start to form intracellularly. At least 26 different human proteins, intact or in fragmented form, are known to form amyloid, which is linked with many debilitating neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and transthyretin (TTR)-related amyloidosis (ATTR). In this work, we focus on ATTR, which is one of the most frequent systemic amyloid diseases. A functional link was established between hereditary ATTR, a severe and fatal disorder and the enhanced propensity of the human plasma protein transthyretin (TTR) to form aggregates, caused by single point mutations in the TTR gene. The disease is heterogeneous and clinical symptoms vary from cardiomyopathy to progressing sensorimotor polyneuropathy depending on TTR variant involved and the amyloid deposition site. Despite the fact that TTR-derived amyloid accumulates in different organs such as heart, kidney, eyes, and predominantly in the peripheral nerves of ATTR patients, the exact mechanism of the disease development is not understood. In contrast to the case of AD, it has been difficult to generate an animal model for ATTR in transgenic mice that would be useful in understanding TTR aggregation processes and the mechanisms of the associated toxicity as these mice did not develop any neuropathic phenotype besides amyloid deposits. Therefore, we created a disease-model in Drosophila due to its huge repertoire of genetic techniques and easy genotype – phenotype translation, as well as its success in modeling human neurodegeneration. We have generated transgenic flies that over-express the clinical amyloidogenic variant TTRL55P, the engineered variant TTR-A (TTRV14N ⁄ V16E), and the wild-type protein. All TTR variants were found in the secreted form in the hemolymph where misfolding occurred and depending on the pool of toxic species, the fate of the fly was decided. Within a few weeks, both mutants (but not the wild-type TTR) demonstrated a time-dependent aggregation of misfolded molecules in vivo. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability, and shortened life span. In contrast, expression of the wild-type TTR had no discernible effect on either longevity or fly behavior. In this work, we also addressed the correlation between TTR transgene dosage and thus, protein levels, with the severity of the phenotypes observed in TTR-A flies which developed a “dragged wing” phenotype. Remarkably, we established that degenerative changes such as damage to the retina strictly correlated with increased levels of mutated TTR but inversely with behavioral alterations and the dragged wing phenotype. We characterized formation of aggregates in the form of 20 nm spherules and amyloid filaments intracellularly in the thoracic adipose tissue and brain glia (both tissues that do not express the transgene). Moreover, we detected a fraction of neurotoxic TTR-A in the hemolymph of young but not old flies. We proposed that these animals counteract formation and persistence of toxic TTR-A species by removal from the circulation into intracellular compartments of glial and fat body cells and this is part of a mechanism that neutralizes the toxic effects of TTR. We validated the fly model for ATTR by applying a genetic screen during study of modifier genes. We found Serum amyloid P component (a product of the APCS gene) as a potent modifier of TTR amyloid-induced toxicity that was effective in preventing the apoptotic response in cell culture assay and capable of reducing the dragged wings when co-expressed in TTR-A flies. Finally, we optimized this fly model in order to screen for therapeutic compounds effective against ATTR. Feeding assays showed the effectiveness of several compounds among known native-state kinetic stabilizers of TTR against its aggregation. We described several early endpoints in this model, which can be used as a rapid and cost-effective method for optimizing concentrations and pre-screening of drug candidates. As the proof of principle, by feeding flies with increasing doses of diflunisal analogue (an FDA-approved Non-Steroidal Anti-Inflammatory Drug) a dose-dependent reduction of the dragged wings was observed.

Killer strains of the yeast Kluyveromyces lactis secrete a heterotrimeric protein toxin (zymocin) to inhibit the growth of sensitive yeasts. The cytotoxicity of zymocin resides in the γ subunit (γ-toxin), however the mechanism of cytotoxicity caused by γ-toxin was previously unknown. This thesis aimed to unravel the mode of γ-toxin action and characterize the interaction between γ-toxin and its substrates. Previous studies suggested a link between the action of γ-toxin and a distinct set of transfer RNAs. In paper I, we show that γ-toxin is a tRNA anticodon endonuclease which cleaves tRNA carrying modified nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at position 34 (wobble position). The cleavage occurs 3’ to the wobble uridine and yields 2’, 3’-cyclic phosphate and 5´-hydroxyl termini. In paper II, we identified the determinants in tRNA important for efficient γ-toxin cleavage. The modifications present on the wobble uridines have different effects on tRNA cleavage by γ-toxin. The Saccharomyces cerevisiae wobble modification mcm5 group has a strong positive effect, whereas the Escherichia coli wobble modification 5-methylaminomethyl group has a strong inhibitory effect on tRNA cleavage. The s2 group present in both S. cerevisiae and E. coli tRNAs has a weaker positive effect on the cleavage. The anticodon stem loop (ASL) of tRNA represents the minimal structural requirement for γ-toxin action. Nucleotides U34U35C36A37C38 in the ASL are required for optimal cleavage by γ-toxin, whereas a purine at position 32 or a G at position 33 dramatically reduces the reactivity of ASL. Screening for S. cerevisiae mutants resistant to zymocin led to the identification of novel proteins important for mcm5s2U formation (paper III). Sit4p (a protein phosphatase), Sap185p and Sap190p (two of the Sit4 associated proteins), and Kti14p (a protein kinase) are required for the formation of mcm5 side chain. Ncs2p, Ncs6p, Urm1p, and Uba4p, the latter two function in a protein modification (urmylation) pathway, are required for the formation of s2 group. The gene product of YOR251C is also involved in the formation of s2 group. The involvement of multiple proteins suggests that the biogenesis of mcm5s2U is very complex.

This thesis describes the target selection, cloning, expression, purification, crystallization, structure and biochemical characterization of five essential Mycobacterium tuberculosis (Mtb) proteins. The search for drugs against the causal agent of tuberculosis is urgently needed and the targeting of essential genes is necessary to fulfill this goal. The crystal structures of carbonic anhydrases (CA) Rv1284 and Rv3588c have been determined to 2.0 and 1.7 Å resolution, respectively. Rv3588c, in contrast to Rv1284, is an active β-CA that shows two different active site conformations and pH-dependent oligomerization states. Rv1295 is an active threonine synthase with an unusually high pH optimum; the structure has been solved to 2.5 Å resolution, based on which a modification to the reaction mechanism published previously is proposed. Mtb has a thick and impermeable cell envelope that constitutes an efficient barrier against drugs. One of the essential components of the envelope is mycolic acid (MA). The inhibition of enzymes participating in its synthesis would be lethal for Mtb. Rv0636, a formerly unknown-function protein has β-hydroxyacyl-ACP dehydrase activity which is essential for MA synthesis. Co-expression with partners notably improves its solubility. Around 55% of Mtb proteins have unknown function. Rv3778c is one of them and its three-dimensional structure has been determined to 1.8 Å resolution. Studies aimed at the elucidation of its biochemical function are shown. A pathway not yet reported in Mtb is also suggested.

Concerns about climate changes and scarcity of fossil fuels are rising. Hence wood is becoming an attractive source of renewable energy and raw material and these new dimensions have prompted increasing interest in wood formation in trees, in both the scientific community and wider public. In this thesis, the focus is on a key process in wood development: programmed cell death (PCD) in the development of xylem elements. Since secondary cell wall formation is dependent, inter alia, upon the life time of xylem elements, the qualitative features of wood will be affected by PCD in xylem, about which there is little information. This thesis focuses on the anatomical, morphological and transcriptional features of PCD during xylem development in both the stem of hybrid aspen, Populus tremula (L.) x tremuloides (Michx.) and the hypocotyl of the herbaceous model system Arabidopsis thaliana (L. Heynh.). In Populus, the progressive removal of organelles from the cytoplasm before the time of death (vacuolar bursts) and the slowness of the cell death process, illustrated by DNA fragmentation assays (such as TUNEL and Comet assays), have been ascertained in the xylem fibres by microscopic analyses. Furthermore, candidate genes for the regulation of fibre cell death were identified either from a Populus EST library obtained from woody tissues undergoing fibre cell death or from microarray experiments in Populus stem, and further assessed in an in silico comparative transcriptomic analysis of Arabidopsis thaliana. These candidate genes were either putative novel regulators of fibre cell death or members of previously described families of cell death-related genes, such as autophagy-related genes. The induction of the latter and the previous microscopic observations suggest the importance of autophagy in the degradation of the cytoplasmic contents specifically in the xylem fibres. Vacuolar bursts in the vessels were the only previously described triggers of PCD in the xylem, which induce the very rapid degradation of the nuclei and surrounding cytoplasmic contents, therefore unravelling a unique previously unrecorded type of PCD in the xylem fibres, principally involving autophagy. Arabidopsis is an attractive alternative model plant for exploring some aspects of wood formation, such as the characterisation of negative regulators of PCD. Therefore, the anatomy of Arabidopsis hypocotyls was also investigated and the ACAULIS5 (ACL5) gene, encoding an enzyme involved in polyamine biosynthesis, was identified as a key regulator of xylem specification, specifically in the vessel elements, though its negative effect on the cell death process. Taken together, PCD in xylem development seems to be a highly specific process, involving unique cell death morphology and molecular machinery. In addition, the technical challenges posed by the complexity of the woody tissues examined highlighted the need for specific methods for assessing PCD and related phenomena in wood. / Oron för klimatförändringar och brist på fossila bränslen har ökat påtagligt under de senaste åren. De enorma möjligheter som skogsråvaran erbjuder som alternativ källa för förnyelsebar energi och råmaterial har väckt ett stort intresse också för den biologiska processen bakom vedbildning i träd. Denna avhandling fokuserar på en viktig process i vedbildning: programmerad celldöd (PCD) i xylemet. Xylemcellernas livstid påverkar bildningen av sekundära cellväggar, vilket i sin tur påverkar vedens kvalitativa egenskaperna, så som veddensitet. Trots dess betydelse för viktiga egenskaper hos vedråvaran existerar fortfarande väldigt lite information om xylem PCD på cellulär eller molekylär nivå. I den här avhandlingen belyses de anatomiska, morfologiska och genetiska aspekterna av PCD i xylemutveckling i både stam av hybridasp, Populus tremula (L.) x tremuloides (Michx.) och hypokotyl av det örtartade modellsystemet Arabidopsis thaliana (L. Heynh.). Xylemet i både Populus och Arabidopsis består av två olika celltyper; de vattentransporterade kärlen och de stödjande fibrerna. Det är känt att celldöd i kärlen pågår mycket snabbt efter att den centrala vakuolen brister och de hydrolytiska enzymer släpps in i cytoplasman. I den här avhandlingen ligger fokus på fibrerna i Populus xylemet. Med hjälp av mikroskopianalyser av cellmorfologin (elektronmikroskopi) och DNA-fragmentering i cellkärnan (TUNEL- och Comet-analyser) kunde vi konstatera att till skillnad från kärlen så uppvisar fibrerna en långsam och progressiv nedbrytning av organellerna och cellkärnans DNA före vakuolbristning. Dessutom har kandidatgener för reglering av fibercelldöd identifierats antingen från ett Populus EST bibliotek från vedartade vävnader som genomgår fibercelldöd eller från mikroarray experiment i Populus stam. Dessa kandidatgener är antingen potentiella nya regulatorer av fibercelldöd eller medlemmar av tidigare beskrivna familjer av celldödsrelaterade gener. Bland de sistnämnda finns autofagi-relaterade gener, vilket stöder funktionen av autofagi i samband med autolys av cellinnehållet i xylemfibrerna. Dessa studier pekar därför på en typ av PCD som har inte tidigare beskrivits för xylemet. Arabidopsis är ett alternativt växtmodellsystem för studier av vissa aspekter av vedbildningen, såsom karakteriseringen av negativa regulatorer av PCD. Därför har också hypokotylanatomin analyserats, och ACAULIS5 (ACL5) genen, som kodar för ett enzym i biosyntesen av polyaminer, har visats vara en viktig regulator av xylemspecifikation genom dess negativa effekt på kärlens celldöd. Sammantaget visar denna avhandling att PCD i xylemutvecklingen verkar involvera unika morfologiska och molekylära mekanismer. Vi visar dessutom att komplexiteten hos de vedartade vävnaderna leder till ett behov av bättre anpassade verktyg för att djupare kunna bedöma PCD och liknande fenomen i veden. / Även med namnet Moreau-Courtois, Charleen L. samt Moreau, Charleen.

The formation of blood, hematopoiesis, is a dynamic process originating from a small number of hematopoietic stem cells (HSCs). To sustain hematopoiesis throughout life HSCs have the unique capacity to differentiate into all mature hematopoietic lineages as well as generating more HSCs by a mechanism referred to as self-renewal. However, the regulation of these processes is largely unknown. During embryonic development HSCs expand in the fetal liver, indicating that this environment supports HSC self-renewal. The LIM-homeobox gene Lhx2 is expressed in the fetal liver during this period and Lhx2 null mutant mice die in utero due to severe anemia caused by an environmental defect in the fetal liver. Embryonic stem cells differentiate in vitro, forming embryoid bodies (EBs) containing various tissues including hematopoietic progenitor cells. Introduction of Lhx2 into this system by retroviral transfer led to the generation of cytokine dependent HSC-like cell lines that were multipotent and expressed surface markers similar to embryonic HSCs. However, the specificity and efficiency of this event could not be elucidated. To further evaluate the function of Lhx2 expression during hematopoietic development, Lhx2 was introduced into an ES cell system where expression could be efficiently turned on. This approach revealed that Lhx2 induce self-renewal of distinct multipotent hematopoietic progenitor/stem cells present in the EB, with the ability to form HSC-like cell lines. The Lhx2 induced self-renewal is growth factor specific since stem cell factor and interleukin-6 are necessary and sufficient for this process. However, Lhx2 expression blocked erythroid differentiation and interfered with early ES cell commitment, indicating that the effect of Lhx2 is cell type specific. Since HSCs of early embryonic origin are inefficient in engrafting adult recipients upon transplantation, we wanted to address whether we could generate cell lines retaining this capacity by expression of Lhx2 in hematopoietic cells from adult bone marrow. This led to the generation of clonal and cytokine dependent HSC-like cell lines capable of generating erythroid, myeloid and lymphoid cells upon transplantation into lethally irradiated recipients. When transplanted into stem cell-deficient mice, they contributed to circulating erythrocytes for at least 18 months, revealing a remarkable potential for self-renewal and differentiation in vivo. However, expression of Lhx2 was maintained in vivo and most engrafted mice developed a transplantable myeloproliferative disorder resembling human chronic myeloid leukemia. Thus, elucidation of the mechanism for Lhx2 function in HSC-like cell lines would give insights into both normal and pathological regulation of HSCs. Down-regulation of Lhx2 expression in HSC-like cell lines with inducible Lhx2 expression led to rapid loss of stem cell characteristics and differentiation into various hematopoietic cell types. Thus, global gene expression analysis comparing Lhx2+ HSC-like cell lines to their Lhx2- progeny would give insights into the molecular basis for Lhx2 function in stem cells. A number of differentially expressed genes overlapped with previously reported HSC enriched genes, further emphasizing the resemblance between HSCs and the HSC-like cell lines also at the molecular level. Moreover, a number of genes were identified with functions or expression patterns related to Lhx2 in other organs. Collectively, these data suggest that these HSC-like cell lines represent a relevant model system for normal HSCs on the molecular and the functional level as well as for evaluating Lhx2 function in the development of various tissues in the embryo as well as in disease.

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci. / QC 20100723

Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates.   Here we describe the investigation of genes and proteins involved in xylan formation as well as the development of a model system that will aid the functional analysis of wood formation. Xylan is the main hemicellulose or cross linking glycan in dicot wood and thereby one of the most abundant carbohydrates on earth. We demonstrate that hybrid aspen cell suspension cultures can be used as a model system for secondary cell wall formation. We have also examined glycosyltransferases from CAZy family 43 that play a part in secondary cell wall formation. We have focused on one of these, Pt×tGT43A, a likely ortholog of Arabidopsis IRX9, which plays a crucial role in xylan formation. The protein was transiently expressed in Nicotiana benthamiana and its function and localization is described. Also, we investigate a glycoside hydrolase, Pt×tXyn10A, involved in wood formation. Its role is not clear but it most likely modifies xylan as it gets incorporated into the secondary cell wall after secretion from the Golgi. This influences the interaction between cellulose, xylan and lignin in the finished wood cell. We have also cloned a transcription factor, Pt×tMYB021, a likely ortholog of Arabidopsis MYB46 and we show that it activates GT43A, GT43B and Xyn10A. By analysis of the promoter sequences we identify a CA-rich motif putatively important for xylem-specific genes.   By mastering proteins involved in xylogenesis we will acquire the tools to improve and develop the wood product market. Xylan is an immense unexploited source of renewable carbohydrate. New products envisioned include e.g. faster growing trees, changed fiber characteristics, optimised utilization of wood carbohydrates for biofuels and biomaterials as well as invention of intelligent materials by biomimetic engineering. / Vedbildning, eller xylogenes, är en grundläggande mekanism för så skilda områden som industri, boende och en hållbar miljö. Ved består av sekundärt xylem som är starka, stora celler med tjocka cellväggar som är lignifierade. Grunden för de starka cellerna är en avancerad komposit bestående av cellulosafibrer tvärbundna av hemicellulosa och slutligen ingjutet i lignin. Denna fiberkomposit är den sekundära cellväggen i vedartade växter. Celldelning och differentiering regleras genom att sätta igång och stänga av gener. Proteiner som kodas av dessa gener utför de viktigaste funktionerna i cellerna. De styr hela maskineriet som upprätthåller cellernas struktur och funktion, underhåller tillväxt samt tillverkar nödvändiga produkter såsom cellväggskolhydraterna. Här beskriver vi utforskningen av gener och proteiner som är inblandade i xylanbildning liksom utvecklandet av ett modellsystem som kommer vara en hjälp i den funktionella analysen av vedbildning. Xylan är den vanligaste hemicellulosan, eller korsbindande glykanen, i lövträd och därför en av de vanligaste kolhydraterna på jorden. Vi demonstrerar att hybridaspcellkulturer i suspension kan användas som ett modellsystem för sekundär cellväggsbildning. Vi har också undersökt glykosyltransferaser från CAZy-familj 43 som tycks spela en viktig roll i bildandet av sekundär cellvägg. Vi har fokuserat på en av dessa, Pt×tGT43A, en trolig ortolog till Arabidopsis IRX9 som spelar en viktig roll i xylanbildning. Proteinet har uttryckts övergående i Nicotiana benthamiana och dess funktion och lokalisering beskrivs. Dessutom undersöker vi ett glykosidhydrolas, Pt×tXyn10A, involverad i vedbildning. Dess roll är oklar men högst sannolikt modifierar det xylan medan det inkorporeras i sekundära cellväggen efter sekretion från Golgi. Detta influerar interaktionen mellan cellulosa, hemicellulosa och lignin i den slutliga vedcellen. Vi har också klonat en transkriptionsfaktor, Pt×tMYB021, en trolig ortolog till Arabidopsis MYB46 och vi visar att den aktiverar GT43A, GT43B och Xyn10A. Genom analys av promotorsekvenserna har vi identifierat ett CA-rikt motiv förmodat viktigt för xylemspecifika gener.Genom att bemästra proteinerna som är ansvariga för vedbildning får vi verktyg att utveckla skogsproduktsmarknaden. Xylan är en ofantligt stor outnyttjad källa till förnyelsebara kolhydrater. En vision är nya produkter som till exempel snabbväxande träd, ändrade fiberegenskaper, optimerat användande av vedkolhydrater för biobränsle och biomaterial såväl som utvecklandet av intelligenta material genom biomimetisk ingenjörskonst. / QC20100730

Transforming growth factor-β (TGF-β) signaling is initiated when the ligand binds to type II and type I serine/threonine kinase receptors at the cell surface. Activated TGF-β type I receptors phosphorylate R-Smads which relocate, together with co-Smads, to the cell nucleus and regulate transcription. Enhancement or repression of Smad-specific gene targets leads to intracellular protein compositions which organize functional complexes and thus govern cellular processes such as proliferation, migration and differentiation. TGF-β/Smad signaling relays are regulated by various post-translational modifications. From receptors to gene promoters, intricate interplays between phosphorylation, acetylation, ubiquitination and numerous other modifications, control Smad signaling initiation and duration. However, many steps in the cascade, including receptor internalization, Smad nuclear shuttling and transcriptional termination, still remain elusive. The open gaps in our understanding of these mechanisms most likely involve additional post-translational regulations. Thus, the aim of the present investigation was to identify novel modulators of TGF-β/Smad signaling. In the first part of this thesis, we show the importance of ADP-ribosylation in Smad-mediated transcription. We identified poly(ADP-ribose) polymerase 1 (PARP-1) as a Smad interacting protein. Our work revealed that PARP-1 forms direct interactions with Smad3/4, and PARylates residues in their MH1 domains. This modification restricts Smads from binding to DNA and attenuates Smad-activated transcription. PARylation is reversed by the glycohydrolase PARG. We provide evidence that PARG can de-ADP-ribosylate Smads, which enhances Smad-promoted gene regulation. In the second part, we examine a Smad-dependent gene target of TGF-β signaling, salt inducible kinase 1 (SIK). After induction, SIK cooperates with Smad7 and Smurf2 to downregulate the TGF-β type I receptor. The mechanism relies on both the kinase and UBA domain of SIK as well as the E3-ligase activity of Smurf2. In summary, we have unveiled two enzyme-dependent TGF-β/Smad modulatory mechanisms; SIK promoted receptor turnover and PARP-1/PARG-regulated Smad signaling.

Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased  replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations.