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  • 1.
    Aili, Margareta
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Role of YopE and LcrH in effector translocation, HeLa cell cytotoxicity and virulence2005Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In order to establish an extra-cellular infection the gram-negative bacteria Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to translocate a set of anti-host effectors into eukaryotic cells. The toxins disrupt signalling pathways important for phagocytosis, cytokine production and cell survival. Secretion and translocation via this T3SS is strictly regulated on several levels. In this context, the function of YopE and LcrH during Yersinia infections has been analysed.

    YopE is an essential translocated effector that disrupts the actin cytoskeleton of infected eukaryotic cells, by inactivating small GTPases through its GTPase activating protein (GAP) activity. However, cytotoxicity can be uncoupled from in vitro GAP activity towards the RhoA, Rac1 and Cdc42 GTPases. Furthermore, in vivo studies of the YopE GAP activity revealed that only RhoA and Rac1 are targeted, but this is not a pre-requisite for Yersinia virulence. Hence, YopE must target one or more additional GTPases to cause disease in mice.

    YopE was the only Yersinia effector that blocks LDH release from infected cells. Moreover, translocated YopE could regulate the level of subsequent effector translocation by a mechanism that involved the YopE GAP function and another T3S component, YopK. Loss of translocation control elevated total T3S gene expression in the presence of eukaryotic cells. This indicated the existence of a regulatory loop for feedback control of T3S gene expression in the bacteria that originates from the interior of the eukaryotic cell after effector translocation is completed. This might represent the true virulence function of YopE.

    Exoenzyme S (ExoS) of Pseudomonas aeruginosa has a YopE-like GAP domain with similar activity towards RhoA, Rac1 and Cdc42. However, ExoS is unable to complement hyper-translocation resulting from loss of YopE. This indicates a unique function for YopE in translocation control in Yersinia that might be dependent on correct intracellular localisation. It follows that the Membrane Localisation Domain in YopE was important for translocation control, but dispensable for cytotoxicity and blockage of LDH release.

    YopD and its cognate chaperone LcrH are negative regulatory elements of the T3S regulon and together with YopB, are involved in the effector translocation process. Randomly generated point mutants in LcrH specifically effected stability and secretion of both the YopB and YopD substrates in vitro and prevented their apparent insertion as translocon pores in the membranes of infected cells. Yet, these mutants still produced stable substrates in the presence of eukaryotic cells and most could mediate at least partial effector translocation. Thus, only minimal amounts of the YopB and YopD translocator proteins are needed for translocation and the LcrH chaperone may regulate this process from inside the bacteria.

  • 2.
    Aili, Margareta
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Telepnev, Max
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Hallberg, Bengt
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Wolf-Watz, Hans
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Rosqvist, Roland
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity.2003Inngår i: Microbial Pathogenesis, ISSN 0882-4010, Vol. 34, nr 6, 297-308 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmid-encoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166–183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell.

  • 3. Aldick, Thomas
    et al.
    Bielaszewska, Martina
    Uhlin, Bernt Eric
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Humpf, Hans-Ulrich
    Wai, Sun Nyunt
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Karch, Helge
    Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin.2009Inngår i: Molecular microbiology, ISSN 1365-2958, Vol. 71, nr 6, 1496-508 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]
    Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.
  • 4.
    Andersson, Karin
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Prefibrillar oligomeric Transthyretin mutants - amyloid conformation, toxicity and association with Serum amyloid P component2005Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Amyloidoses represent a heterogeneous group of diseases characterized by abnormal protein metabolism leading to extracellular deposition of fibrillar, proteinaceous amyloid in various tissues and organs of the body. To date more than 20 different proteins have been linked to diseases with amyloid depositions, of which Alzheimer’s disease and the prion-associated diseases are the most well known. Despite the origin of protein in the amyloid, the fibrils share some common biochemical and biophysical properties such as a diameter of 8-13 nm, a β-pleated sheet secondary structure packed in an ordered crystal-like way, Congo red and thioflavin binding with characteristic spectroscopic patterns and decoration of the fibrils with Serum amyloid P component and glycoseaminoglycans.

    The plasma protein transthyretin (TTR) is associated with familial amyloidosis with polyneuropathy (FAP) and senile systemic amyloidosis (SSA). FAP is a lethal, autosomal inherited disorder caused by point mutations in the TTR-gene. More than 80 different mutations have been associated with amyloid formation and linked to FAP. The interpretation is that amino acid replacements at different sites of the polypeptide lead to reduced stability. Mutant TTR were constructed that have a strong tendency to self-aggregate under physiological conditions. The precipitates were shown to be amyloid by staining with thioflavin T and Congo red. As the mutants were sensitive to trypsin cleavage compared to plasma TTR, we suggest that the mutants represent amyloid precursors or that they may share structural properties with intermediates on a pathway leading to amyloid deposition. Monoclonal antibodies were generated that exclusively recognize the amyloidogenic folding of TTR providing direct biochemical evidence for a structural change in amyloidogenic intermediates. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and is proposed to be displaced at the initial phase of amyloid formation. Amyloidogenic intermediates of TTR were shown to induce a toxic, free radical dependent, response in cultured neuroblastoma cells. Morphological studies revealed a correlation between toxicity (apoptosis) and the presence of immature amyloid suggesting that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.

    Serum amyloid P component (SAP) is a highly conserved plasma glycoprotein universally found associated with amyloid depositions independently of protein origin. SAP’s role in amyloid formation is contradictory since both inhibition and promotion of aggregation have been shown in the case of fibril formation from the Aβ peptide of Alzheimer’s disease. Amyloidogenic prefibrils of TTR were shown to bind SAP and no interference with aggregation was detected. SAP co-localize in patches with mutant TTR on the surface of neuroblastoma cells and prevent apoptosis induced by mutant TTR and Aβ peptide, while several other molecules known to decorate amyloid fibrils were without effect.

  • 5.
    Andersson, Karin
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Lundgren, Erik
    Inhibition of amyloid induced apoptosis - a new role for Serum amyloid P componentManuskript (Annet vitenskapelig)
  • 6.
    Andersson, Karin
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Olofsson, Anders
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Holm Nielsen, Ellen
    Svehag, SvenErik
    Lundgren, Erik
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Only amyloidogenic inermediates of transthyretin induce apoptosis2002Inngår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, Vol. 294, nr 2, 309-314 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In diseases like Alzheimer's disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism. (c) 2002 Elsevier Science (USA).

  • 7.
    Antonsson, Åsa
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Regulation of NF-κB by Calmodulin2003Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Cells experience numerous external signals which they must respond to. Such signals arriving at the cell surface are transduced via various signal transduction pathways and often ultimately result in regulation of transcription. NF-κB is a family of transcription factors involved in the regulation of genes important for processes such as immune and inflammatory responses, cell growth, development and cell survival. NF-κB proteins are normally kept inactive in the cytoplasm due to masking of their nuclear localisation signal (NLS) by inhibitory IκB proteins. A large number of stimuli lead to the activation of IκB-kinase (IKK). Active IKK phosphorylates IκB and thereby labels it for ubiquitination and, subsequently, degradation by the proteasome. Liberated NF-κB enters the nucleus, where it takes part in the regulation of its target genes.

    Calmodulin (CaM) is a ubiquitous Ca2+-binding protein which is considered to be the predominant intracellular Ca2+ sensor. CaM plays a major role in the Ca2+-dependent regulation of a wide variety of cellular processes, including transcription. CaM regulates transcription both indirectly through CaM-dependent kinases and phosphatases and directly through interaction with transcription factors.

    CaM was found to bind directly and in a Ca2+-dependent fashion to the two NF-κB family members c-Rel and RelA. The CaM-NF-κB interactions were strongly enhanced by NF-κB activating stimuli and this enhancement was blocked by the addition of IκB, suggesting that c-Rel and RelA can bind CaM after their signal-induced release from IκB. Compared to wild-type c-Rel, CaM binding-deficient mutants were shown to exhibit an increased nuclear accumulation and transcriptional activity on Ca2+-regulated cytokine promoters. The results suggest that CaM can inhibit transport of c-Rel, but not of RelA, to the nucleus and thereby differentially regulate the activation of NF-κB proteins following cell stimulation. CaM was also found to affect NF-κB activity indirectly through the action of a CaM-dependent kinase (CaMK). Studies of the events leading to IκBα phosphorylation revealed that CaM and CaMKII inhibitors blocked phorbol ester induced activation of IKK. Furthermore, CaM and CaMKII inhibitors also blocked T cell receptor/CD3 induced IκBα degradation, and expression of an inhibitor-resistant derivative of the γ isoform of CaMKII caused the inhibitors lose their effect on phorbol ester induced IκBα degradation. Finally, expression of a constitutively active CaMKII resulted in the activation of NF-κB. These results identify CaMKII as a mediator of IKK activation, specifically in response to T cell receptor/CD3 and phorbol ester stimulation.

    In conclusion, this thesis describes the identification of CaM as a dual regulator of NF-κB proteins, acting both directly and indirectly to affect the activity of this family of transcription factors.

  • 8.
    Aspholm-Hurtig, Marina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Dailide, Giedrius
    Lahmann, Martina
    Kalia, Awdhesh
    Ilver, Dag
    Roche, Niamh
    Vikström, Susanne
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Sjöström, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lindén, Sara
    Bäckström, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Lundberg, Carina
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mahdavi, Jafar
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Nilsson, Ulf J
    Velapatiño, Billie
    Gilman, Robert H
    Gerhard, Markus
    Alarcon, Teresa
    López-Brea, Manuel
    Nakazawa, Teruko
    Fox, James G
    Correa, Pelayo
    Dominguez-Bello, Maria Gloria
    Perez-Perez, Guillermo I
    Blaser, Martin J
    Normark, Staffan
    Carlstedt, Ingemar
    Oscarson, Stefan
    Teneberg, Susann
    Berg, Douglas E
    Borén, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin.2004Inngår i: Science, ISSN 0036-8075, Vol. 305, nr 5683, 519-522 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.

  • 9.
    Bailey, Leslie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gylfe, Asa
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sundin, Charlotta
    Muschiol, Sandra
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Nordström, Peter
    Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin. Umeå universitet, Medicinska fakulteten, Institutionen för samhällsmedicin och rehabilitering, Geriatrik.
    Henriques-Normark, Birgitta
    Lugert, Raimond
    Waldenström, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle2007Inngår i: FEBS Lett, ISSN 0014-5793, Vol. 581, nr 4, 587-595 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.

  • 10.
    Berg, A. H.
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Olsson, P-E.
    17ß-estradiol induced vitellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char (Salvelinus alpinus).Manuskript (Annet vitenskapelig)
  • 11.
    Berg, A. H.
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Thomas, P.
    Olsson, P-E.
    Characterization of the 17,20β-dihydroxy-4-pregnen-3-one membrane receptor in Arctic char (Salvelinus alpinus) ovaries and its upregulation during gonadotropin induction of oocyte maturation.Manuskript (Annet vitenskapelig)
  • 12.
    Berg, Håkan
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Teleost reproduction: Aspects of Arctic char (Salvelinus alpinus) oocyte growth and maturation.2003Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems.

    All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels.

    It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char.

    In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors.

    Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation

  • 13.
    Bergqvist, Ingela
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Eriksson, Maria
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Eriksson, Björn
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Corneliussen, Brit
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development2000Inngår i: European Journal of Immunology, ISSN 0014-2980, Vol. 30, nr 10, 2857-2863 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    E2A, HEB and E2-2 genes encode a group of basic helix-loop-helix (bHLH) transcription factors that are structurally and functionally similar. Deletion of the genes encoding either of these proteins leads to early lethality and a block in B lymphocyte development. Evidence for a function in T lymphocyte development has, however, only been reported for E2A and HEB. To further elucidate the role of E2-2 at developmental stages that have proven difficult to study due to the early lethality phenotype of mice defective in E2-2, we generated and analyzed mice conditionally mutated in the E2-2 gene. These mice are mosaic with respect to E2-2 expression, consisting of cells with either one functional and one null mutated E2-2 allele or two null mutated alleles. Using this experimental model, we find that cells with a homozygous null mutated E2-2 gene are under-represented in B lymphocyte as well as T lymphocyte cell lineages as compared to other hematopoietic or non-hematopoietic cell lineages. Our data suggests that E2-2 deficiency leads to a partial block in both B and T lymphocyte development. The block in T cell development appears to occur at an early stage in differentiation, since skewing in the mosaicism is observed already in CD4+8+ double-positive thymocytes.

  • 14.
    Bernardo, Lisandro
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    On the role of ppGpp and DksA mediated control of σ54-dependent transcription2006Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

  • 15.
    Bernardo, Lisandro
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Johansson, Linda
    Solera, Dafne
    Skärfstad, Eleonore
    Szalewska-Palasz, Agnieszka
    Shingler, Victoria
    The role of the alarmone ppGpp and DksA in regulation of σ54-dependent transcription in Pseudomonas putidaManuskript (Annet vitenskapelig)
  • 16.
    Birve, A
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Chen, S.
    Rasmuson-Lestander, Å.
    Expression pattern of the Drosophila polycomb group gene Suppressor of zeste 12.Manuskript (Annet vitenskapelig)
  • 17.
    Birve, A.
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Chen, S.
    Rasmuson-Lestander, Å.
    Suppressor of zeste12 mediates silencing through PREs, interacts genetically with other PcG genes and and has a unique binding pattern on polytene chromosomes.Manuskript (Annet vitenskapelig)
  • 18.
    Birve, Anna
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Suppressor of zeste 12, a Polycomb group gene in Drosophila melanogaster; one piece in the epigenetic puzzle2003Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing Drosophila embryo, the proper expression pattern of e.g. the homeotic genes Abd-B and Ubx is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression.

    As part of this thesis, Suppressor of zeste 12 (Su(z)12) is characterized as a Drosophila Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes Abd-B and Ubx, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. Su(z)12 mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the Su(z)12 gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. Su(z)12 was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between Su(z)12 and zeste. I also show that Su(z)12 interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3.

    In conclusion, results presented in this thesis show that the recently identified Pc-G gene, Su(z)12, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes.

  • 19.
    Bröms, Jeanette
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Type III secretion- the various functions of the translocon operon in bacterial pathogenesis2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In order to establish colonisation of a human host, pathogenic Yersinia use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the lcrGVHyopBD translocon operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous pcrGVHpopBD operon of P. aeruginosa as a comparative tool.

    Yersinia spp. and P. aeruginosa were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in Yersinia that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded.

    The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in Yersinia, while equivalent complexes with analogous proteins were not formed in P. aeruginosa. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to Yersinia.

    Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in P. aeruginosa. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.

  • 20.
    Bröms, Jeanette
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Edqvist, Petra
    Forsberg, Åke
    Francis, Matthew
    Mapping of an YscY binding regulatory domain within the type III secretion chaperone LcrH of Yersinia pseudotuberculosis.Manuskript (Annet vitenskapelig)
  • 21.
    Bröms, Jeanette
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Forslund, Anna-Lena
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis.2003Inngår i: Microbiology, ISSN 1350-0872, Microbiology, Vol. 149, nr 9, 2615-2626 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The homologous pcrGVHpopBD and lcrGVHyopBD translocase operons of Pseudomonas aeruginosa and pathogenic Yersinia spp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. In Yersinia, this operon is also required for yop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability of popB/yopB and popD/yopD was investigated. Secretion of PopB produced in trans in a yopB null mutant of Yersinia was only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement the yopB translocation defect. The Yersinia yopD null mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependent yop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functional Yersinia type III secretion.

  • 22.
    Bröms, Jeanette
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Forslund, Anna-Lena
    Forsberg, Åke
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Francis, Matthew
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    PcrH of Pseudomonas aeruginosa is essential for secretion and assembly of the type III translocon2003Inngår i: Journal of Infectious Diseases, ISSN 0022-1899, Vol. 188, nr 12, 1909-1921 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pseudomonas aeruginosa harbors a type III secretion system that translocates antihost effectors into an infected eukaryotic cell. PcrH is a key component of type III secretion in this essential virulence strategy. In the absence of PcrH, P. aeruginosa is translocation deficient because of a specific reduction in presecretory stability and subsequent secretion of PopB and PopD, 2 proteins essential for the translocation process. PcrH exerts this chaperone function by binding directly to PopB and PopD. Consistent with the genetic relatedness of PcrH with LcrH of pathogenic Yersinia species, these proteins are functionally interchangeable with respect to their ability to complement the translocation defect associated with either a lcrH or pcrH null mutant, respectively. Thus, the translocator class of chaperones performs a critical function in ensuring the assembly of a translocation competent type III secreton. Finally, unlike the regulatory roles of other translocator-class chaperones (e.g., LcrH, SicA of Salmonella enterica, and IpgC of Shigella species), in vitro regulation of P. aeruginosa type III secretion does not involve PcrH.

  • 23.
    Bröms, Jeanette
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Sundin, Charlotta
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Francis, Matthew
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Comparative analysis of type III effector translocation by Yersinia pseudotuberculosis expressing native LcrV or PcrV from Pseudomonas aeruginosa.2003Inngår i: Journal of Infectious Diseases, ISSN 0022-1899, Vol. 188, nr 2, 239-249 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The homologues LcrV of Yersinia species and PcrV of Pseudomonas aeruginosa are pore-forming components. When expressed in a Yersinia lcrV background, PcrV formed smaller pores in infected erythrocyte membranes, correlating to a lowered translocation of Yersinia effectors. To understand this phenomenon, cytotoxins exoenzyme S of P. aeruginosa and YopE of Yersinia were introduced into a Yersinia background without Yop effectors but expressing LcrV or PcrV. Comparable translocation of each substrate indicated that substrate recognition by LcrV/PcrV is not a regulator of translocation. Yersinia harboring pcrV coexpressed with its native operon efficiently translocated effectors into HeLa cell monolayers and formed large LcrV-like pores in erythrocyte membranes. Thus, a PcrV complex with native P. aeruginosa translocon components is required to form fully functional pores for complete complementation of effector translocation in Yersinia.

  • 24.
    Bylund, Göran O.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lövgren, J. Mattias
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Wikström, P. Mikael
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Characterization of mutations in the metY-nusA-infB operon that suppress the slow growth of a DeltarimM mutant.2001Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, nr 20, 6095-6106 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits. The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA. Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a DeltarimM mutant by increasing the expression of rbfA. Nine of these mutations were in the nusA gene, which is located upstream from rbfA in the metY-nusA-infB operon; three mutations deleted the transcriptional terminator between infB and rbfA; one was an insertion of IS2 in infB, creating a new promoter for rbfA; and one was a duplication, placing a second copy of rbfA downstream from a promoter for the yhbM gene. Two of the nusA mutations were identical, while another mutation (nusA98) was identical to a previously isolated mutation, nusA11, shown to decrease termination of transcription. The different nusA mutations were found to increase the expression of rbfA by increasing the read-through of two internal transcriptional terminators located just downstream from the metY gene and that of the internal terminator preceding rbfA. Induced expression of the nusA(+) gene from a plasmid in a nusA(+) strain decreased the read-through of the two terminators just downstream from metY, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the metY-nusA-infB operon expression is these terminators. All of the nusA mutations produced temperature-sensitive phenotypes of rimM(+) strains. The nusA gene has previously been shown to be essential at 42 degrees C and below 32 degrees C. Here, we show that nusA is also essential at 37 degrees C.

  • 25.
    Byström, Anders S
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Björk, Glenn R
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The structural gene (trmD) for the tRNA(m1G)methyltransferase is part of a four polypeptide operon in Escherichia coli K-12.1982Inngår i: Molecular General Genetics, ISSN 0026-8925, Vol. 188, nr 3, 447-454 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The trmD gene, which is the structural gene for the tRNA(m1G)-methyltransferase, is shown to be part of a polycistronic operon. A 4.6 kb SalI-EcoRI chromosomal DNA fragment contains the trmD gene (Byström and Björk 1982). Subclonings, deletion mapping and Tn5 insertions into plasmid pBY03 have established the gene organization of the trmD area on the Escherichia coli chromosome. The different plasmid derivatives were analysed for expression of protein products using the minicell system. Such analyses established the organisation of genes encoding six polypeptides to be SalI1-48 K-13 K-25 K-31 K-15 K-16 K-EcoRI1. The 31 K polypeptide was shown to be the tRNA(m1G)methyltransferase. The trmD operon encodes for four polypeptides; 13 K-25 K-31 K(trmD)-15 K and the direction of transcription is from 13 K (promoter proximal) to 15 K (promoter distal). However, there might be a weak internal promoter between the trmD gene and the gene encoding the 15 K product. The level of expression from this operon in the minicell system does not seem to follow normal polarity since we observed high expression of 13 K, 25 K, and 15 K products but low expression of the internal trmD gene.

  • 26.
    Byström, Anders S
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fink, G R
    A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast1989Inngår i: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 216, nr 2-3, 276-286 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA(IMet). Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA(IMet) is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slow; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.

  • 27.
    Byström, Anders S
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hjalmarsson, Karin J
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Wikström, P Mikael
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Björk, Glenn R
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide1983Inngår i: EMBO Journal, ISSN 0261-4189, Vol. 2, nr 6, 899-905 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.

  • 28.
    Byström, Anders S
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    von Gabain, A
    Björk, Glenn R
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species.1989Inngår i: Journal of Molecular Biology, ISSN 0022-2836, Vol. 208, nr 4, 575-586 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively. The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.

  • 29.
    Carlsson, Anna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Forsgren, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Neurologi.
    Nylander, P-O
    Hellman, Urban
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Forsman-Semb, Kristina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Holmgren, Gösta
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Holmberg, Monica
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Identification of a susceptibility locus for migraine with and without aura on 6p12.2-p21.1.2002Inngår i: Neurology, ISSN 0028-3878, Vol. 59, nr 11, 1804-1807 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 30.
    Carlsson, Lennart
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Aspects of interferon alpha signalling in hematopoetic cells2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment.

    We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002.

    In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes.

    The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.

  • 31.
    Carlsson, Lennart
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Dacklin, Ingrid
    Persson, Håkan
    Golovleva, Irina
    Ruuth, Kristina
    Lundgren, Erik
    Characterisation of IFN resistance in a myeloma cell line with an unstable genomeManuskript (preprint) (Annet vitenskapelig)
  • 32.
    Carlsson, Lennart
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Ruuth, Kristina
    Lundgren, Erik
    IFN-alpha induced proliferation of human primary B-lymphocytesManuskript (preprint) (Annet vitenskapelig)
  • 33.
    Chapman, K B
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Byström, Anders S
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Boeke, J D
    Initiator methionine tRNA is essential for Ty1 transposition in yeast1992Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 89, nr 8, 3236-3240 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.

  • 34.
    Chen, Peng
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar Typhimurium2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described.

    A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C.

    cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent.

    During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

  • 35.
    Chen, Peng
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Näsvall, S. Joakim
    Björk, Glenn R.
    Uridine-5-oxyacetic acid (cmo5U) present in the wobble position of a subset of tRNAs in Salmonella enterica Serovar Typhimurium has a tRNA dependent influence on coding capacity and cell physiologyManuskript (Annet vitenskapelig)
  • 36.
    Chen, Peng
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Qian, Qiang
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Zhao, Shaoping
    Isaksson, Leif A.
    Björk, Glenn R.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine2002Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 317, nr 4, 481-492 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA. Copyright 2002 Elsevier Science Ltd.

  • 37.
    Chen, Sa
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Larsson, Anna L.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Tegeling, Erik
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Birve, Anna
    Umeå universitet, Medicinsk fakultet, Medicinsk biovetenskap.
    Rasmuson-Lestander, Åsa
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    In vivo analysis of Suppressor of zeste 12´s different isoformsManuskript (Annet vitenskapelig)
    Abstract [en]

    Polycomb Group (PcG) genes are known to encode a large chromatin-associated family of proteins which are involved in genomic regulation of many cellular processes. Su(z)12 is a key component in PcG silencing. It is needed for three levels of methylation of histone 3 lysine 27 in vivo in Drosophila. Here, we report that Su(z)12 may exist in different isoforms and that these isoforms are spatially and temporally regulated. The biological function of the Su(z)12-A and -B isoforms seems to be very different. For instance the transgenic Su(z)12-B and the human homolog SUZ12, but not Su(z)12-A, rescue Su(z)12 mutants. Furthermore, transgenic flies over-expressing Su(z)12-B show typical homeotic transformation phenotypes, while over-expression of Su(z)12-A does not. However, the two isoforms appears to be able to substitute for each other in some aspects. During larval and pupal stages, Su(z)12-A seems to play the main role. 

  • 38.
    Chen, Sa
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Rasmuson-Lestander, Åsa
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Regulation of the Drosophila engrailed gene by Polycomb repressor complex 22009Inngår i: Mechanisms of Development, ISSN 0925-4773, E-ISSN 1872-6356, Vol. 126, nr 5-6, 443-448 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Suppressor-of-zeste-12 (Su(z)12) is a core component of the Polycomb repressive complex 2 (PRC2), which has a methyltransferase activity directed towards lysine residues of histone 3. Mutations in Polycomb group (PcG) genes cause de-repression of homeotic genes and subsequent homeotic transformations. Another target for Polycomb silencing is the engrailed gene, which encodes a key regulator of segmentation in the early Drosophila embryo. In close proximity to the en gene is a Polycomb Response Element, but whether en is regulated by Su(z)12 is not known. In this report, we show that en is not de-repressed in Su(z)12 or Enhancer-of-zeste mutant clones in the anterior compartment of wing discs. Instead, we find that en expression is down-regulated in the posterior portion of wing discs, indicating that the PRC2 complex acts as an activator of en. Our results indicate that this is due to secondary effects, probably caused by ectopic expression of Ubx and Abd-B.

  • 39.
    Chen, Sa
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Rasmuson-Lestander, Åsa
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    The role of Suppressor of zeste 12 in cell cycle regulationManuskript (Annet vitenskapelig)
    Abstract [en]

    Polycomb group (PcG) proteins control a large amount of target genes and are essential for genomic programming and differentiation. Many members in the PcG family have been shown to be upregulated in different types of cancers. Suppressor of zeste 12 (Su(z)12) is an essential component in PcG silencing and is necessary for histone 3 lysine 27 tri-methylation in vivo. To unravel a possible role of Su(z)12 in cell cycle regulation, we first investigate the localization pattern of Su(z)12 in Drosophila wildtype testes and embryos by immunohistochemical staining. We found that Su(z)12 was dynamically regulated during cell division. Further investigation of the function of Su(z)12 in cell division was done by cell number counting, apoptosis and proliferation marker staining in Su(z)12 somatic knockout clones in wing discs. The conclusion from the small wing phenotype in Su(z)12 knockout wing discs is that Su(z)12 may increase apoptosis and decrease cell proliferation rate.

  • 40.
    Croxatto, Antony
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    VanT, a central regulator of quorum sensing signalling in Vibrio anguillarum2006Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Many bacteria produce signal molecules that serve in a cell-to-cell communication system termed quorum sensing. This signalling system allows a bacterial population to co-ordinately regulate functions according to their cell number in a defined environment. As bacterial growth progresses towards the stationary phase, signalling molecules accumulate in the growth medium and, above a certain threshold level, regulate the expression of genes involved in diverse functions. Most of the functions monitored by quorum sensing are most beneficial when they are performed as a population than by single cells, such as virulence factor production, biofilm formation, conjugation and bioluminescence.

    Vibrio anguillarum is a bacterial pathogen that causes terminal hemorrhagic septicaemia in marine fish. V. anguillarum possesses multiple quorum sensing circuits similar to the LuxI/LuxR and the V. harveyi-type systems. In this study, a characterisation of the quorum sensing-regulated transcriptional activator VanT was made. VanT belongs to the V. harveyi LuxR family of transcriptional regulators, which play a central role in quorum sensing signalling in Vibrio species. VanT was shown to regulate serine, metalloprotease, pigment, exopolysaccharide (EPS) and biofilm production. VanT repressed an EPS locus that plays a critical role in bacterial colonization of the fish integument and virulence.

    The V. harveyi-like quorum sensing systems were shown to limit rather than induce vanT expression throughout growth in V. anguillarum. In contrast to homologous proteins in other Vibrio spp., the quorum sensing phosphorelay protein VanU and the response regulator VanO had antagonistic roles in the regulation of vanT expression. Unlike other members of the luxR family, vanT was expressed at low cell density and no significant induction due to quorum sensing regulation was seen.

    Interestingly, VanT expression was induced by the alternative sigma factor RpoS as the cells entered stationary phase. RpoS was shown to regulate VanT expression post-transcriptionally by promoting vanT mRNA stability. VanT and RpoS were important for bacterial survival under stress conditions, indicating that VanT is likely an essential factor of V. anguillarum stress response.

  • 41.
    Croxatto, Antony
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Chalker, Victoria J
    Lauritz, Johan
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Jass, Jana
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Hardman, Andrea
    Williams, Paul
    Cámara, Miguel
    Milton, Debra L
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    VanT, a Homologue of Vibrio harveyi LuxR, Regulates Serine, Metalloprotease, Pigment, and Biofilm Production in Vibrio anguillarum2002Inngår i: Journal of Bacteriology, ISSN 0021-9193, Vol. 184, nr 6, 1617-1629 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract
  • 42.
    Croxatto, Antony
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Pride, John
    Hardman, Andrea
    Williams, Paul
    Cámara, Miguel
    Milton, Debra L
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A distinctive dual-channel quorum-sensing system operates in Vibrio anguillarum.2004Inngår i: Molecular Microbiology, ISSN 0950-382X, Vol. 52, nr 6, 1677-1689 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing. In marine Vibrio species, the Vibrio harveyi-type LuxR protein is a key player in a quorum-sensing phosphorelay cascade, which controls the expression of virulence, symbiotic and survival genes. Previously, we characterized Vibrio anguillarum homologues of LuxR (VanT) and LuxMN (VanMN) and, in this study, we have identified homologues of LuxPQ (VanPQ) and LuxOU (VanOU). In contrast to other Vibrio species, vanT was expressed at low cell density and showed no significant induction as the cell number increased. In addition, although the loss of VanO increased vanT expression, the loss of VanU, unexpectedly, decreased it. Both VanN and VanQ were required for repression of vanT even in a vanU mutant, suggesting an alternative route for VanNQ signal transduction other than via VanU. VanT negatively regulated its own expression by binding and repressing the vanT promoter and by binding and activating the vanOU promoter. The signal relay results in a cellular response as expression of the metalloprotease, empA, was altered similar to that of vanT in all the mutants. Consequently, the V. anguillarum quorum-sensing phosphorelay systems work differently from those of V. harveyi and may be used to limit rather than induce vanT expression.

  • 43.
    Croxatto, Antony
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Weber, Barbara
    Chen, Chang
    Milton, Debra L
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Post-transcriptional regulation of the Vibrio anguillarum quorum-sensing regulator vanT by RpoSManuskript (Annet vitenskapelig)
  • 44.
    Deleuil, Fabienne
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Mogemark, Lena
    Umeå universitet, Medicinska fakulteten, Molekylärbiologi (Teknat- och Medfak).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism2003Inngår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 5, nr 1, 53-64 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.

  • 45. Dever, Thomas E
    et al.
    Yang, Weimin
    Åström, Stefan
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Byström, Anders S
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hinnebusch, A G
    Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes.1995Inngår i: Molecular and Cellular Biology, ISSN 0270-7306, Vol. 15, nr 11, 6351-6363 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA(iMet), reduced gene dosage of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA(iMet) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2.GTP. Met-tRNA(iMet) ternary complex is the principal component limiting translation in cells when eIF-2 alpha is phosphorylated on serine 51. Analysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2-GTP. Met-tRNA(iMet) ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.

  • 46. Edqvist, Petra
    et al.
    Aili, Margareta
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Francis, Matthew
    Minimal secretion of the YopB and YopD translocators is sufficient for Yop-effector translocation by Yersinia.Manuskript (Annet vitenskapelig)
  • 47. Edqvist, Petra
    et al.
    Bröms, Jeanette
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Steggo, Peter
    Forsberg, Åke
    Francis, Matthew
    Characterization of the tetratricopeptide repeats in type III secretion chaperones- mediators of substrate binding and specificity.Manuskript (Annet vitenskapelig)
  • 48.
    Edqvist, Petra J
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis2007Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior.

    The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors.

    Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation.

    Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.

  • 49.
    Edqvist, Petra J
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Bröms, Jeanette E
    Åhlund, Monika K
    Forsberg, Åke
    Francis, Matthew S
    Functional insights into the YopD C-terminus through comprehensive site-directed mutagenesisManuskript (Annet vitenskapelig)
  • 50.
    Edqvist, Petra J
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Francis, Matthew S
    Examination of LcrH type III secretion chaperone function during Yersinia-eukaryotic cell contactManuskript (Annet vitenskapelig)
12345 1 - 50 of 207
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