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Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.
Barbara Michalec-Wawiórka; Jakub Czapiński; Kamil Filipek; Patrycja Rulak; Arkadiusz Czerwonka; Marek Tchórzewski; Adolfo Rivero-Müller. An Improved Vector System for Homogeneous and Stable Gene Regulation. International Journal of Molecular Sciences 2021, 22, 5206 .
AMA StyleBarbara Michalec-Wawiórka, Jakub Czapiński, Kamil Filipek, Patrycja Rulak, Arkadiusz Czerwonka, Marek Tchórzewski, Adolfo Rivero-Müller. An Improved Vector System for Homogeneous and Stable Gene Regulation. International Journal of Molecular Sciences. 2021; 22 (10):5206.
Chicago/Turabian StyleBarbara Michalec-Wawiórka; Jakub Czapiński; Kamil Filipek; Patrycja Rulak; Arkadiusz Czerwonka; Marek Tchórzewski; Adolfo Rivero-Müller. 2021. "An Improved Vector System for Homogeneous and Stable Gene Regulation." International Journal of Molecular Sciences 22, no. 10: 5206.
Aging is a biological phenomenon common to all living organisms. It is thought that the rate of aging is influenced by diverse factors, in many cases related to the control of energy metabolism, i.e., the so-called pro-longevity effects of starvation. Translation, regarded as the main energy consumption process, lies at the center of interest, as it has a significant impact on the longevity phenomenon. It has been shown that perturbations in the translational apparatus may lead to a lower rate of aging. Therefore, the main aim of this study was to investigate aging in relation to the protein biosynthesis circuit, taking into account the uL11 ribosomal protein as a vital ribosomal element. To this end, we used set of yeast mutants with deleted single uL11A or uL11B genes and a double disruptant uL11AB mutant. We applied an integrated approach analyzing a broad range of biological parameters of yeast mutant cells, especially the longevity phenomenon, supplemented with biochemical and high throughput transcriptomic and metobolomic approaches. The analysis showed that the longevity phenomenon is not fully related to the commonly considered energy restriction effect, thus the slow-down of translation does not represent the sole source of aging. Additionally, we showed that uL11 can be classified as a moonlighting protein with extra-ribosomal function having cell-cycle regulatory potential.
Mateusz Mołoń; Eliza Molestak; Monika Kula-Maximenko; Przemysław Grela; Marek Tchórzewski. Ribosomal Protein uL11 as a Regulator of Metabolic Circuits Related to Aging and Cell Cycle. Cells 2020, 9, 1745 .
AMA StyleMateusz Mołoń, Eliza Molestak, Monika Kula-Maximenko, Przemysław Grela, Marek Tchórzewski. Ribosomal Protein uL11 as a Regulator of Metabolic Circuits Related to Aging and Cell Cycle. Cells. 2020; 9 (7):1745.
Chicago/Turabian StyleMateusz Mołoń; Eliza Molestak; Monika Kula-Maximenko; Przemysław Grela; Marek Tchórzewski. 2020. "Ribosomal Protein uL11 as a Regulator of Metabolic Circuits Related to Aging and Cell Cycle." Cells 9, no. 7: 1745.
The uL10‐protein is the main constituent of the ribosomal‐P‐stalk, anchoring the whole stalk to the ribosome through interactions with rRNA. The P‐stalk is the core of the GTPase associated center (GAC), a critical element for ribosome biogenesis and ribosome translational activity. All P‐stalk proteins (uL10, P1 and P2) undergo phosphorylation within their C‐termini. Here, we show that uL10 has multiple phosphorylation sites, mapped also within the N‐terminal rRNA‐binding‐domain. Our results reveal that the introduction of a negative charge within the N‐terminus of uL10 impairs its association with the ribosome. These findings demonstrate that uL10 N‐terminal phosphorylation has regulatory potential governing the uL10 interaction with the ribosome and may control the activity of GAC.
Kamil Filipek; Barbara Michalec‐Wawiórka; Aleksandra Boguszewska; Sebastian Kmiecik; Marek Tchórzewski. Phosphorylation of the N‐terminal domain of ribosomal P‐stalk protein uL10 governs its association with the ribosome. FEBS Letters 2020, 594, 3002 -3019.
AMA StyleKamil Filipek, Barbara Michalec‐Wawiórka, Aleksandra Boguszewska, Sebastian Kmiecik, Marek Tchórzewski. Phosphorylation of the N‐terminal domain of ribosomal P‐stalk protein uL10 governs its association with the ribosome. FEBS Letters. 2020; 594 (18):3002-3019.
Chicago/Turabian StyleKamil Filipek; Barbara Michalec‐Wawiórka; Aleksandra Boguszewska; Sebastian Kmiecik; Marek Tchórzewski. 2020. "Phosphorylation of the N‐terminal domain of ribosomal P‐stalk protein uL10 governs its association with the ribosome." FEBS Letters 594, no. 18: 3002-3019.
Malaria remains one the most infectious and destructive protozoan diseases worldwide.Plasmodium falciparum, a protozoan parasite with a complex life cycle and high genetic variability responsible for the difficulties in vaccine development, is implicated in most malaria-related deaths. In the course of study, we prepared a set of antigens based on P-proteins fromP. falciparumand determined their immunogenicity in anin vivoassay on a mouse model. The pentameric complex P0-(P1-P2)2was prepared along with individual P1, P2, and P0 antigens. We determined the level of cellular- and humoral-type immunological response followed by development of specific immunological memory. We have shown that the number of Tc cells increased significantly after the first immunization with P2 and after the second immunization with P1 and P0-(P1-P2)2, which highly correlated with the number of Th1 cells. P0 appeared as a poor inducer of cellular response. After the third boost with P1, P2, or P0-(P1-P2)2, the initially high cellular response dropped to the control level accompanied by elevation of the number of activated Treg cells and a high level of suppressive TGF-β. Subsequently, the humoral response against the examined antigens was activated. Although the titers of specific IgG were increasing during the course of immunization for all antigens used, P2 and P0-(P1-P2)2were found to be significantly stronger than P1 and P0. A positive correlation between the Th2 cell abundance and the level of IL-10 was observed exclusively after immunization with P0-(P1-P2)2. Anin vitroexposure of spleen lymphocytes from the immunized mice especially to the P1, P2, and P0-(P1-P2)2protein caused 2-3-fold higher cell proliferation than that in the case of lymphocytes from the nonimmunized animals, suggesting development of immune memory. Our results demonstrate for the first time that the native-like P-protein pentameric complex represents much stronger immune potential than individual P-antigens.
Agnieszka Szuster-Ciesielska; Leszek Wawiórka; Dawid Krokowski; Nikodem Grankowski; Łukasz Jarosz; Urszula Lisiecka; Marek Tchórzewski. Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)2ofPlasmodium falciparumin a Mouse Model. Journal of Immunology Research 2019, 2019, 1 -19.
AMA StyleAgnieszka Szuster-Ciesielska, Leszek Wawiórka, Dawid Krokowski, Nikodem Grankowski, Łukasz Jarosz, Urszula Lisiecka, Marek Tchórzewski. Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)2ofPlasmodium falciparumin a Mouse Model. Journal of Immunology Research. 2019; 2019 ():1-19.
Chicago/Turabian StyleAgnieszka Szuster-Ciesielska; Leszek Wawiórka; Dawid Krokowski; Nikodem Grankowski; Łukasz Jarosz; Urszula Lisiecka; Marek Tchórzewski. 2019. "Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)2ofPlasmodium falciparumin a Mouse Model." Journal of Immunology Research 2019, no. : 1-19.
The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the sarcin-ricin-loop (SRL) on the 60S ribosomal subunit is a major source of its toxicity. The model proposes that SRL depurination leads to protein synthesis inhibition, evoking ribotoxic stress with concomitant induction of numerous metabolic pathways, which lead to cell death. However, the direct relationship between the depurination and its impact on the translational machinery in vivo has never been satisfactorily explained. In this work, we approached a long-standing question about the influence of SRL depurination on the functioning of the translational machinery in vivo. We have shown that an already low level of depurinated ribosomes exert an effect on cell metabolism, indicating that minute modification within the ribosomal pool is sufficient to elicit a toxic effect. Importantly, depurination does not affect notably any particular step of translation, and translational slowdown caused by ricin is not a direct consequence of depurination and cannot be considered as the sole source of cell death. Instead, SRL depurination in a small fraction of ribosomes blocks cell cycle progression with no effect on cell viability. In this work, we have provided a comprehensive picture of the impact of SRL depurination on the translational apparatus in vivo. We propose that ribosomes with depurinated SRL represent a small imprinted ribosomal pool, which generates a specific signal for the cell to halt the cell cycle.
Monika Szajwaj; Leszek Wawiórka; Eliza Molestak; Barbara Michalec-Wawiórka; Mateusz Mołoń; Iwona Wojda; Marek Tchórzewski. The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2019, 1866, 118554 .
AMA StyleMonika Szajwaj, Leszek Wawiórka, Eliza Molestak, Barbara Michalec-Wawiórka, Mateusz Mołoń, Iwona Wojda, Marek Tchórzewski. The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2019; 1866 (12):118554.
Chicago/Turabian StyleMonika Szajwaj; Leszek Wawiórka; Eliza Molestak; Barbara Michalec-Wawiórka; Mateusz Mołoń; Iwona Wojda; Marek Tchórzewski. 2019. "The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity." Biochimica et Biophysica Acta (BBA) - Bioenergetics 1866, no. 12: 118554.
The genome-wide duplication event observed in eukaryotes represents an interesting biological phenomenon, extending the biological capacity of the genome at the expense of the same genetic material. For example, most ribosomal proteins in Saccharomyces cerevisiae are encoded by a pair of paralogous genes. It is thought that gene duplication may contribute to heterogeneity of the translational machinery; however, the exact biological function of this event has not been clarified. In this study, we have investigated the functional impact of one of the duplicated ribosomal proteins, uL6, on the translational apparatus together with its consequences for aging of yeast cells. Our data show that uL6 is not required for cell survival, although lack of this protein decreases the rate of growth and inhibits budding. The uL6 protein is critical for the efficient assembly of the ribosome 60S subunit, and the two uL6 isoforms most likely serve the same function, playing an important role in the adaptation of translational machinery performance to the metabolic needs of the cell. The deletion of a single uL6 gene significantly extends the lifespan but only in cells with a high metabolic rate. We conclude that the maintenance of two copies of the uL6 gene enables the cell to cope with the high demands for effective ribosome synthesis.
Lidia Borkiewicz; Mateusz Mołoń; Eliza Molestak; Przemysław Grela; Patrycja Horbowicz-Drożdżal; Leszek Wawiórka; Marek Tchórzewski. Functional Analysis of the Ribosomal uL6 Protein of Saccharomyces cerevisiae. Cells 2019, 8, 718 .
AMA StyleLidia Borkiewicz, Mateusz Mołoń, Eliza Molestak, Przemysław Grela, Patrycja Horbowicz-Drożdżal, Leszek Wawiórka, Marek Tchórzewski. Functional Analysis of the Ribosomal uL6 Protein of Saccharomyces cerevisiae. Cells. 2019; 8 (7):718.
Chicago/Turabian StyleLidia Borkiewicz; Mateusz Mołoń; Eliza Molestak; Przemysław Grela; Patrycja Horbowicz-Drożdżal; Leszek Wawiórka; Marek Tchórzewski. 2019. "Functional Analysis of the Ribosomal uL6 Protein of Saccharomyces cerevisiae." Cells 8, no. 7: 718.
Ricin belongs to the group of ribosome-inactivating proteins (RIPs), i.e., toxins that have evolved to provide particular species with an advantage over other competitors in nature. Ricin possesses RNA N-glycosidase activity enabling the toxin to eliminate a single adenine base from the sarcin-ricin RNA loop (SRL), which is a highly conserved structure present on the large ribosomal subunit in all species from the three domains of life. The SRL belongs to the GTPase associated center (GAC), i.e., a ribosomal element involved in conferring unidirectional trajectory for the translational apparatus at the expense of GTP hydrolysis by translational GTPases (trGTPases). The SRL represents a critical element in the GAC, being the main triggering factor of GTP hydrolysis by trGTPases. Enzymatic removal of a single adenine base at the tip of SRL by ricin blocks GTP hydrolysis and, at the same time, impedes functioning of the translational machinery. Here, we discuss the consequences of SRL depurination by ricin for ribosomal performance, with emphasis on the mechanistic model overview of the SRL modus operandi.
Przemysław Grela; Monika Szajwaj; Patrycja Horbowicz-Drożdżal; Marek Tchórzewski. How Ricin Damages the Ribosome. Toxins 2019, 11, 241 .
AMA StylePrzemysław Grela, Monika Szajwaj, Patrycja Horbowicz-Drożdżal, Marek Tchórzewski. How Ricin Damages the Ribosome. Toxins. 2019; 11 (5):241.
Chicago/Turabian StylePrzemysław Grela; Monika Szajwaj; Patrycja Horbowicz-Drożdżal; Marek Tchórzewski. 2019. "How Ricin Damages the Ribosome." Toxins 11, no. 5: 241.
Staphylococcus epidermidis small colony variants can survive inside macrophages and their survival has been proposed as a pivotal process in the pathogenesis of biomaterial associated infections. In the present study the intracellular location of clinical isolates of SCV and parental wild type strains inside macrophages was determined. Furthermore, the effect of IFN-γ and rapamycin on the level of SCV/WT as well as lysosomes colocalisation and iNOS induction in THP-activated macrophages in response to WT and SCV strains of Staphylococcus epidermidis were examined. It was demonstrated that SCV strain of S. epidermidis can survive and persist inside macrophages and its intracellular survival is supported by the induction of phagosomal acidification. The ability to reduce the high proportion of LysoTracker positive SCV containing phagosomes was exclusively found when IFN-γ was used. The findings suggest that IFN-γ mediates SCV killing via two distinct mechanisms, phagosome alkalisation and an increased iNOS synthesis, so the cytokine may control S. epidermidis WT and SCV infection in macrophages. Staphylococcus epidermidis SCV is a less potent stimulus of iNOS than the WT strain and the feature may help SCV to persist in hostile environment of macrophages. Rapamycin treatment did not influence the iNOS synthesis but reduced the percentage of both bacterial strains within acidic organelles. However, the percentage of SCV within LysoTracker positive organelles, even though reduced comparing to non-primed cells, was higher than in the WT strain indicating that Staphylococcus epidermidis possesses unique metabolic features allowing SCV to survive within macrophages.
Agnieszka Magryś; Kamil Derylo; Agnieszka Bogut; Alina Olender; Marek Tchórzewski. Intraphagolysosomal conditions predispose to Staphylococcus epidermidis small colony variants persistence in macrophages. PLOS ONE 2018, 13, e0207312 .
AMA StyleAgnieszka Magryś, Kamil Derylo, Agnieszka Bogut, Alina Olender, Marek Tchórzewski. Intraphagolysosomal conditions predispose to Staphylococcus epidermidis small colony variants persistence in macrophages. PLOS ONE. 2018; 13 (11):e0207312.
Chicago/Turabian StyleAgnieszka Magryś; Kamil Derylo; Agnieszka Bogut; Alina Olender; Marek Tchórzewski. 2018. "Intraphagolysosomal conditions predispose to Staphylococcus epidermidis small colony variants persistence in macrophages." PLOS ONE 13, no. 11: e0207312.
Although a lot of effort has been put into the search for factors responsible for aging in yeast mother cells, our knowledge of cellular changes in daughter cells originating from old mothers is still very limited. It has been shown that an old mother is not able to compensate for all negative changes within its cell and therefore transfers them to the bud. In this paper, we show for the first time that daughter cells of an old mother have a reset lifespan expressed in units of time despite drastic reduction of their budding lifespan, which suggests that a single yeast cell has a fixed programmed longevity regardless of the time point at which it was originated. Moreover, in our study we found that longevity parameters are not correlated with the rDNA level, DNA damage, chromosome structure or aging parameters (budding lifespan and total lifespan).
Mateusz Molon; Anita Panek; Eliza Molestak; Marek Skoneczny; Marek Tchórzewski; Maciej Wnuk. Daughters of the budding yeast from old mothers have shorter replicative lifespans but not total lifespans. Are DNA damage and rDNA instability the factors that determine longevity? Cell Cycle 2018, 17, 1173 -1187.
AMA StyleMateusz Molon, Anita Panek, Eliza Molestak, Marek Skoneczny, Marek Tchórzewski, Maciej Wnuk. Daughters of the budding yeast from old mothers have shorter replicative lifespans but not total lifespans. Are DNA damage and rDNA instability the factors that determine longevity? Cell Cycle. 2018; 17 (10):1173-1187.
Chicago/Turabian StyleMateusz Molon; Anita Panek; Eliza Molestak; Marek Skoneczny; Marek Tchórzewski; Maciej Wnuk. 2018. "Daughters of the budding yeast from old mothers have shorter replicative lifespans but not total lifespans. Are DNA damage and rDNA instability the factors that determine longevity?" Cell Cycle 17, no. 10: 1173-1187.
The ribosomal uL10 protein, formerly known as P0, is an essential element of the ribosomal GTPase-associated center responsible for the interplay with translational factors during various stages of protein synthesis. In eukaryotic cells, uL10 binds two P1/P2 protein heterodimers to form a pentameric P-stalk, described as uL10-(P1-P2)2, which represents the functional form of these proteins on translating ribosomes. Unlike most ribosomal proteins, which are incorporated into pre-ribosomal particles during early steps of ribosome biogenesis in the nucleus, P-stalk proteins are attached to the 60S subunit in the cytoplasm. Although the primary role of the P-stalk is related to the process of translation, other extraribosomal functions of its constituents have been proposed, especially for the uL10 protein; however, the list of its activities beyond the ribosome is still an open question. Here, by the combination of biochemical and advanced fluorescence microscopy techniques, we demonstrate that upon nucleolar stress induction the uL10 protein accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein. Importantly, using a novel approach, FRAP-AC (FRAP after photoConversion), we have shown that the ribosome-free pool of uL10 represents a population of proteins released from pre-existing ribosomes. Taken together, our data indicate that the presence of uL10 on the ribosomes is affected in stressed cells, thus it might be considered as a regulatory element responding to environmental fluctuations.
Kamil Derylo; Barbara Michalec-Wawiórka; Dawid Krokowski; Leszek Wawiórka; Maria Hatzoglou; Marek Tchórzewski. The uL10 protein, a component of the ribosomal P-stalk, is released from the ribosome in nucleolar stress. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2018, 1865, 34 -47.
AMA StyleKamil Derylo, Barbara Michalec-Wawiórka, Dawid Krokowski, Leszek Wawiórka, Maria Hatzoglou, Marek Tchórzewski. The uL10 protein, a component of the ribosomal P-stalk, is released from the ribosome in nucleolar stress. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2018; 1865 (1):34-47.
Chicago/Turabian StyleKamil Derylo; Barbara Michalec-Wawiórka; Dawid Krokowski; Leszek Wawiórka; Maria Hatzoglou; Marek Tchórzewski. 2018. "The uL10 protein, a component of the ribosomal P-stalk, is released from the ribosome in nucleolar stress." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1865, no. 1: 34-47.
Frataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussed
Mostafa Fekry; Wessen Alshokry; Przemysław Grela; Marek Tchórzewski; Eva-Christina Ahlgren; Christopher A. Söderberg; Oleksandr Gakh; Grazia Isaya; Salam Al-Karadaghi. SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY. PLOS ONE 2017, 12, e0184961 .
AMA StyleMostafa Fekry, Wessen Alshokry, Przemysław Grela, Marek Tchórzewski, Eva-Christina Ahlgren, Christopher A. Söderberg, Oleksandr Gakh, Grazia Isaya, Salam Al-Karadaghi. SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY. PLOS ONE. 2017; 12 (9):e0184961.
Chicago/Turabian StyleMostafa Fekry; Wessen Alshokry; Przemysław Grela; Marek Tchórzewski; Eva-Christina Ahlgren; Christopher A. Söderberg; Oleksandr Gakh; Grazia Isaya; Salam Al-Karadaghi. 2017. "SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY." PLOS ONE 12, no. 9: e0184961.
The P-stalk represents a vital element within the ribosomal GTPase-associated center, which represents a landing platform for translational GTPases. The eukaryotic P-stalk exists as a uL10-(P1-P2) 2 pentameric complex, which contains five identical C-terminal domains, one within each protein, and the presence of only one such element is sufficient to stimulate factor-dependent GTP hydrolysis in vitro and to sustain cell viability. The functional contribution of the P-stalk to the performance of the translational machinery in vivo , especially the role of P-protein multiplication, has never been explored. Here, we show that ribosomes depleted of P1/P2 proteins exhibit reduced translation fidelity at elongation and termination steps. The elevated rate of the decoding error is inversely correlated with the number of the P-proteins present on the ribosome. Unexpectedly, the lack of P1/P2 has little effect in vivo on the efficiency of other translational GTPase (trGTPase)-dependent steps of protein synthesis, including translocation. We have shown that loss of accuracy of decoding caused by P1/P2 depletion is the major cause of translation slowdown, which in turn affects the metabolic fitness of the yeast cell. We postulate that the multiplication of P-proteins is functionally coupled with the qualitative aspect of ribosome action, i.e., the recoding phenomenon shaping the cellular proteome.
Leszek Wawiórka; Eliza Molestak; Monika Szajwaj; Barbara Michalec-Wawiórka; Mateusz Mołoń; Lidia Borkiewicz; Przemysław Grela; Aleksandra Boguszewska; Marek Tchórzewski. Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation. Molecular and Cellular Biology 2017, 37, e00060-17 .
AMA StyleLeszek Wawiórka, Eliza Molestak, Monika Szajwaj, Barbara Michalec-Wawiórka, Mateusz Mołoń, Lidia Borkiewicz, Przemysław Grela, Aleksandra Boguszewska, Marek Tchórzewski. Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation. Molecular and Cellular Biology. 2017; 37 (17):e00060-17.
Chicago/Turabian StyleLeszek Wawiórka; Eliza Molestak; Monika Szajwaj; Barbara Michalec-Wawiórka; Mateusz Mołoń; Lidia Borkiewicz; Przemysław Grela; Aleksandra Boguszewska; Marek Tchórzewski. 2017. "Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation." Molecular and Cellular Biology 37, no. 17: e00060-17.
Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX–PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.
Monika Marek-Kozaczuk; Sylwia Wdowiak-Wrobel; Michał Kalita; Mykhaylo Chernetskyy; Kamil Derylo; Marek Tchórzewski; Anna Skorupska. Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens. Antonie van Leeuwenhoek 2017, 110, 1729 -1744.
AMA StyleMonika Marek-Kozaczuk, Sylwia Wdowiak-Wrobel, Michał Kalita, Mykhaylo Chernetskyy, Kamil Derylo, Marek Tchórzewski, Anna Skorupska. Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens. Antonie van Leeuwenhoek. 2017; 110 (12):1729-1744.
Chicago/Turabian StyleMonika Marek-Kozaczuk; Sylwia Wdowiak-Wrobel; Michał Kalita; Mykhaylo Chernetskyy; Kamil Derylo; Marek Tchórzewski; Anna Skorupska. 2017. "Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens." Antonie van Leeuwenhoek 110, no. 12: 1729-1744.
The eukaryotic P-stalk contains two P1-P2 protein dimers with a conserved C- terminal domain (CTD) critical for the interaction with external factors. To understand the role of the individual CTD of human P1/P2 proteins, we examined the interaction of reconstituted human P-protein complexes and C-terminally truncated forms with ricin A chain (RTA), which binds to the stalk to depurinate the sarcin/ricin loop (SRL). The interaction between P-protein complexes and RTA was examined by surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis and bio-layer interferometry. The P1-P2 heterodimer missing a CTD on P2 was able to bind RTA. In contrast, the P1-P2 heterodimer missing the CTD of P1 protein displayed almost no binding toward RTA. Very low interaction was detected between RTA and the non-truncated P2-P2 homodimer, suggesting that the structural architecture of the P1-P2 heterodimer is critical for binding RTA. The reconstituted pentameric human stalk complex had higher affinity for RTA than the P1-P2 dimer. Deletion of P1 CTD, but not P2 CTD reduced the affinity of the pentamer for RTA. These results highlight the importance of the heterodimeric organization of P1-P2 in the human stalk pentamer and functional non-equivalence of the individual P-protein CTDs in the interaction with RTA.
Przemysław Grela; Xiao-Ping Li; Patrycja Horbowicz; Monika Dźwierzyńska; Marek Tchórzewski; Nilgun E. Tumer. Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus. Scientific Reports 2017, 7, 5608 .
AMA StylePrzemysław Grela, Xiao-Ping Li, Patrycja Horbowicz, Monika Dźwierzyńska, Marek Tchórzewski, Nilgun E. Tumer. Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus. Scientific Reports. 2017; 7 (1):5608.
Chicago/Turabian StylePrzemysław Grela; Xiao-Ping Li; Patrycja Horbowicz; Monika Dźwierzyńska; Marek Tchórzewski; Nilgun E. Tumer. 2017. "Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus." Scientific Reports 7, no. 1: 5608.
The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and β-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.
Aneta Sowa-Jasiłek; Agnieszka Zdybicka-Barabas; Sylwia Stączek; Jerzy Wydrych; Krzysztof Skrzypiec; Paweł Mak; Kamil Derylo; Marek Tchórzewski; Małgorzata Cytryńska. Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells. Microbiological Research 2016, 193, 121 -131.
AMA StyleAneta Sowa-Jasiłek, Agnieszka Zdybicka-Barabas, Sylwia Stączek, Jerzy Wydrych, Krzysztof Skrzypiec, Paweł Mak, Kamil Derylo, Marek Tchórzewski, Małgorzata Cytryńska. Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells. Microbiological Research. 2016; 193 ():121-131.
Chicago/Turabian StyleAneta Sowa-Jasiłek; Agnieszka Zdybicka-Barabas; Sylwia Stączek; Jerzy Wydrych; Krzysztof Skrzypiec; Paweł Mak; Kamil Derylo; Marek Tchórzewski; Małgorzata Cytryńska. 2016. "Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells." Microbiological Research 193, no. : 121-131.
Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.
Kamila Rachwał; Aleksandra Boguszewska; Joanna Kopcińska; Magdalena Karaś; Marek Tchórzewski; Monika Janczarek. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover. Frontiers in Microbiology 2016, 7, 1302 .
AMA StyleKamila Rachwał, Aleksandra Boguszewska, Joanna Kopcińska, Magdalena Karaś, Marek Tchórzewski, Monika Janczarek. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover. Frontiers in Microbiology. 2016; 7 ():1302.
Chicago/Turabian StyleKamila Rachwał; Aleksandra Boguszewska; Joanna Kopcińska; Magdalena Karaś; Marek Tchórzewski; Monika Janczarek. 2016. "The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover." Frontiers in Microbiology 7, no. : 1302.
The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of ‘ribosomal life’. uL11 is involved in ‘birth’ (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the ‘active life’ of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase ‘productive state’ stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.
Leszek Wawiórka; Eliza Molestak; Monika Szajwaj; Barbara Michalec-Wawiórka; Aleksandra Boguszewska; Lidia Borkiewicz; Vladyslava Liudkovska; Joanna Kufel; Marek Tchórzewski. Functional analysis of the uL11 protein impact on translational machinery. Cell Cycle 2016, 15, 1060 -1072.
AMA StyleLeszek Wawiórka, Eliza Molestak, Monika Szajwaj, Barbara Michalec-Wawiórka, Aleksandra Boguszewska, Lidia Borkiewicz, Vladyslava Liudkovska, Joanna Kufel, Marek Tchórzewski. Functional analysis of the uL11 protein impact on translational machinery. Cell Cycle. 2016; 15 (8):1060-1072.
Chicago/Turabian StyleLeszek Wawiórka; Eliza Molestak; Monika Szajwaj; Barbara Michalec-Wawiórka; Aleksandra Boguszewska; Lidia Borkiewicz; Vladyslava Liudkovska; Joanna Kufel; Marek Tchórzewski. 2016. "Functional analysis of the uL11 protein impact on translational machinery." Cell Cycle 15, no. 8: 1060-1072.
Despite many controversies, the yeast Saccharomyces cerevisiae continues to be used as a model organism for the study of aging. Numerous theories and hypotheses have been created for several decades, yet basic mechanisms of aging have remained unclear. Therefore, the principal aim of this work is to propose a possible mechanism leading to increased longevity in yeast. In this paper, we suggest for the first time that there is a link between decreased metabolic activity, fertility and longevity expressed as time of life in yeast. Determination of reproductive potential and total lifespan with the use of fob1Δ and sfp1Δ mutants allows us to compare the “longevity” presented as the number of produced daughters with the longevity expressed as the time of life. The results of analyses presented in this paper suggest the need for a change in the definition of longevity of yeast by taking into consideration the time parameter. The mutants that have been described as “long-lived” in the literature, such as the fob1Δ mutant, have an increased reproductive potential but live no longer than their standard counterparts. On the other hand, the sfp1Δ mutant and the wild-type strain produce a similar number of daughter cells, but the former lives much longer. Our results demonstrate a correlation between the decreased efficiency of the translational apparatus and the longevity of the sfp1Δ mutant. We suggest that a possible factor regulating the lifespan is the rate of cell metabolism. To measure the basic metabolism of the yeast cells, we used the isothermal microcalorimetry method. In the case of sfp1Δ, the flow of energy, ATP concentration, polysome profile and translational fitness are significantly lower in comparison with the wild-type strain and the fob1Δ mutant.
Mateusz Molon; Monika Szajwaj; Marek Tchórzewski; Andrzej Skoczowski; Ewa Niewiadomska; Renata Zadrag-Tecza. The rate of metabolism as a factor determining longevity of the Saccharomyces cerevisiae yeast. AGE 2016, 38, 11 .
AMA StyleMateusz Molon, Monika Szajwaj, Marek Tchórzewski, Andrzej Skoczowski, Ewa Niewiadomska, Renata Zadrag-Tecza. The rate of metabolism as a factor determining longevity of the Saccharomyces cerevisiae yeast. AGE. 2016; 38 (1):11.
Chicago/Turabian StyleMateusz Molon; Monika Szajwaj; Marek Tchórzewski; Andrzej Skoczowski; Ewa Niewiadomska; Renata Zadrag-Tecza. 2016. "The rate of metabolism as a factor determining longevity of the Saccharomyces cerevisiae yeast." AGE 38, no. 1: 11.
Protein Mrt4 is one of trans-acting factors involved in ribosome biogenesis, which in higher eukaryotic cells contains a C-terminal extension similar to the C-terminal part of ribosomal P proteins. We show that human Mrt4 (hMrt4/MRTO4) undergoes phosphorylation in vivo and that serines S229, S233, and S235, placed within its acidic C-termini, have been phosphorylated by CK2 kinase in vitro. Such modification does not alter the subcellular distribution of hMrt4 in standard conditions but affects its molecular behavior during ActD induced nucleolar stress. Thus, we propose a new regulatory element important for the stress response pathway connecting ribosome biogenesis with cellular metabolism.
Barbara Michalec-Wawiorka; Leszek Wawiorka; Kamil Derylo; Dawid Krokowski; Aleksandra Boguszewska; Eliza Molestak; Monika Szajwaj; Marek Tchorzewski. Molecular behavior of human Mrt4 protein, MRTO4, in stress conditions is regulated by its C-terminal region. The International Journal of Biochemistry & Cell Biology 2015, 69, 233 -240.
AMA StyleBarbara Michalec-Wawiorka, Leszek Wawiorka, Kamil Derylo, Dawid Krokowski, Aleksandra Boguszewska, Eliza Molestak, Monika Szajwaj, Marek Tchorzewski. Molecular behavior of human Mrt4 protein, MRTO4, in stress conditions is regulated by its C-terminal region. The International Journal of Biochemistry & Cell Biology. 2015; 69 ():233-240.
Chicago/Turabian StyleBarbara Michalec-Wawiorka; Leszek Wawiorka; Kamil Derylo; Dawid Krokowski; Aleksandra Boguszewska; Eliza Molestak; Monika Szajwaj; Marek Tchorzewski. 2015. "Molecular behavior of human Mrt4 protein, MRTO4, in stress conditions is regulated by its C-terminal region." The International Journal of Biochemistry & Cell Biology 69, no. : 233-240.
The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins. We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods. All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)2 form represents the most favorable architecture for parasite P-proteins. The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate. On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.
Leszek Wawiórka; Dawid Krokowski; Yuliya Gordiyenko; Daniel Krowarsch; Carol Robinson; Ishag Adam; Nikodem Grankowski; Marek Tchórzewski. In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates. Biochimica et Biophysica Acta (BBA) - General Subjects 2014, 1850, 150 -158.
AMA StyleLeszek Wawiórka, Dawid Krokowski, Yuliya Gordiyenko, Daniel Krowarsch, Carol Robinson, Ishag Adam, Nikodem Grankowski, Marek Tchórzewski. In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates. Biochimica et Biophysica Acta (BBA) - General Subjects. 2014; 1850 (1):150-158.
Chicago/Turabian StyleLeszek Wawiórka; Dawid Krokowski; Yuliya Gordiyenko; Daniel Krowarsch; Carol Robinson; Ishag Adam; Nikodem Grankowski; Marek Tchórzewski. 2014. "In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates." Biochimica et Biophysica Acta (BBA) - General Subjects 1850, no. 1: 150-158.