Konzervovaný mechanismus cílení Tail-anchored proteinů u eukaryot

Membránové proteiny, které tvoří přibližně jednu čtvrtinu všech proteinů v buňce, jsou transportovány skrze hydrofilní prostředí cytosolu, než jsou integrovány do membrány organel, nebo do plasmatické membrány. Jedná se o složitý proces, který vyžaduje správné načasování i souhru řady zúčastněných proteinů. Skupina transmembránových proteinů, tzv. Tail-anchored (TA) proteinů, neobsahuje klasický signální peptid umístěný na N-konci proteinu, který proteiny cílí k membráně endoplasmatického retikula. Tato informace je místo toho obsažena v jediné transmembránové doméně na C-konci TA proteinů. Z tohoto důvodu musí být TA proteiny transportovány až po ukončení translace. Hlavní způsob transportu TA proteinů je zprostředkován tzv. Guided Entry of Tail-anchored protein drahou neboli GET drahou. Tuto dráhu tvoří u kvasinek celkem šest (Sgt2, Get1-Get5) a u člověka sedm (navíc Bag6) proteinů, které mají za úkol rozeznat a navázat TA protein, přenést ho k membráně endoplazmatického retikula a zprostředkovat jeho vložení do membrány. Kromě modelových systémů, byly některé z proteinů GET dráhy studovány u rostlin a u Plasmodium falciparum. To do značné míry komplikuje naše pochopení obecného mechanismu dráhy i její využití napříč eukaryoty. Rozhodli jsem se proto charakterizovat GET dráhu u parazitického...Approximately one-fourth of all cellular proteins represent integral membrane proteins (IMPs) that are transported through the cytosol across or into the organellar or plasma membrane. Transport of IMPs requires precise timing which needs to be precisely regulated for them to reach their final destination. Tail-anchored (TA) proteins represent specific class of membrane proteins that lack the N-terminal signal peptide, which targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for the co-translational transport. Instead, they possess single C-terminal transmembrane domain (TMD) that serves as their targeting signal. Therefore, TA proteins are transported only post-translationally when the C-terminal TMD appears from the ribosome. The Guided Entry of Tail-anchored proteins (GET) pathway is the dominant way of how TA proteins find their way into the ER membrane. It is a multistep process that is mediated by six (Sgt2, Get1-Get5) proteins in yeast and seven (plus Bag6) proteins in human, which involves recognition of a TA protein, its targeting to the ER membrane and the actual membrane insertion. In addition to the model cell systems, some of GET pathway components were studied in plants and recently in Plasmodium falciparum, which makes our knowledge on the distribution and the...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes

Conserved mechanism for targeting of Tail-anchored proteins in eukaryotes

Approximately one-fourth of all cellular proteins represent integral membrane proteins (IMPs) that are transported through the cytosol across or into the organellar or plasma membrane. Transport of IMPs requires precise timing which needs to be precisely regulated for them to reach their final destination. Tail-anchored (TA) proteins represent specific class of membrane proteins that lack the N-terminal signal peptide, which targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for the co-translational transport. Instead, they possess single C-terminal transmembrane domain (TMD) that serves as their targeting signal. Therefore, TA proteins are transported only post-translationally when the C-terminal TMD appears from the ribosome. The Guided Entry of Tail-anchored proteins (GET) pathway is the dominant way of how TA proteins find their way into the ER membrane. It is a multistep process that is mediated by six (Sgt2, Get1-Get5) proteins in yeast and seven (plus Bag6) proteins in human, which involves recognition of a TA protein, its targeting to the ER membrane and the actual membrane insertion. In addition to the model cell systems, some of GET pathway components were studied in plants and recently in Plasmodium falciparum, which makes our knowledge on the distribution and the..

Cellular Protein Transport and Its Role in Patogenesis

Předmětem této práce jsou procesy sekrece vybraných proteinů u několika významných parazitů člověka - Toxoplasma gongii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp. a Giardia intestinalis. Uvedeny jsou zde proteiny, jak parazitární, tak i hostitelské a to především v souvislosti s hlavními infekčními procesy a ve spojitosti se sekreční dráhou. Sekrece proteinů do prostředí hostitele patří zejména pro intracelulární parazity mezi klíčové nástroje pro přežití a manipulaci s hostitelským organismem. Z uvedených informací vyplývá, že různí parazité k tomu využívají funkčně i evolučně odlišné strategie. Klíčová slova sekreční dráha, translokon, signální sekvence, Toxoplasma gongii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp., Giardia intestinalisThe main topic of this thesis are the protein secretion processes in several important human parasites - Toxoplasma gondii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp. and Giardia intestinalis. Described here are the parasite's and the host proteins which participate in the pathogenic processes involving the protein secretion. As shown here, the protein secretion into the host environment is one of key tools serving the parasite to survive within and manipulate the host organism. Interestingly, different parasitic organisms use functionally and evolutionary distinct strategies to fulfill this aim. Key words secretory pathway, translocon, signal sequence, Toxoplasma gongii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp., Giardia intestinalisKatedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

The role of untranslated mRNA regions in Giardia intestinalis.

Giardia intestinalis is an anaerobic protozoan pathogen, agent of the disease known as giardiasis. The regulation of gene expression during giardia cell- and life-cycle has been poorly studied so far, with the exception of variable surface proteins, which constitute the immunoprotective coat of the cell. In this diploma thesis, we focus on the possible role of the 3' untranslated region (3'UTR) of mRNA that mediate stability and localization of mRNA transcripts. We use RNA binding proteins of PUF family, which control the function of the target transcripts by their repression, activation or sequestration, to monitor and verify the role of 3'UTRs. These only eukaryotic proteins are highly evolutionarily conserved. Each of them contain highly conserved C-terminal domain, which specificly binds to 3'UTR of mRNAs. We have identified five different PUF proteins in the genome of G. intestinalis (GiPUF), cinfirmed their expression in G. intestinalis trophozoites and located all five proteins in the cytoplasm. GiPUF2, GiPUF3 and GiPUF5 show an additional affinity to the surface of the endoplasmic reticulum. We have identified the C-terminal binding domain in protein sequences of all GiPUF. The most conserved GiPUF4 contain eight binding sites, nearly identical to the binding site of human Pum1 protein,..

Cellular Protein Transport and Its Role in Patogenesis

The main topic of this thesis are the protein secretion processes in several important human parasites - Toxoplasma gondii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp. and Giardia intestinalis. Described here are the parasite's and the host proteins which participate in the pathogenic processes involving the protein secretion. As shown here, the protein secretion into the host environment is one of key tools serving the parasite to survive within and manipulate the host organism. Interestingly, different parasitic organisms use functionally and evolutionary distinct strategies to fulfill this aim. Key words secretory pathway, translocon, signal sequence, Toxoplasma gongii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp., Giardia intestinali

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis

Expression of HaloTagged proteins in <i>G. intestinalis</i> and <i>T. vaginalis</i>.

<p>Western blot analyses of cellular fractions of <i>G. intestinalis</i> and <i>T. vaginalis</i> transformants expressing GiIscU-Halo and TvFtx-Halo fusions, respectively. A) GiIscU-Halo was detected by specific anti-IscU polyclonal antibodies in cell lysate and high-speed pellet (HSP). Two bands in these fractions represent the nuclear encoded (GiIscU) and episomally encoded HaloTag fusion (GiIscU-Halo). B) TvFtx-Halo product was detected by anti-HA monoclonal antibodies in <i>T. vaginalis</i> cellular fractions. The fusion protein was found exclusively in cell lysate and in hydrogenosomes. The upper panels demonstrate the protein profile on the coomassie stained SDS-PAGE gel. Lys-lysate, Cyt-cytosol, HSP-high-speed pellet, Hyd-hydrogenosomes.</p

Mitosomal and hydrogenosomal localization of HaloTagged proteins.

<p>Immunofluorescence analyses of <i>G. intestinalis</i> and <i>T. vaginalis</i> transformants expressing GiIscU-Halo and TvFtx-Halo fusion, respectively. Cells were incubated with TMR-Halo ligand (red), washed and fixed for immunofluorescence analysis. A) TMR-Halo labeled <i>G. intestinalis</i> cells were fixed and labeled by anti-Tom40 specific polyclonal antibodies (green). B) TMR-Halo labeled <i>T. vaginalis</i> cells were fixed and decorated by anti-malic enzyme specific polyclonal antibodies (green). Nuclei were stained with DAPI (blue).</p

Live imaging of mitosomes and hydrogenosomes.

<p>Halo-TMR labeled organelles were followed in living cells. A) Labeled <i>G. intestinalis</i> cells were allowed to attach to the bottom of the well and directly observed while B) the labeled <i>T. vaginalis</i> cells were mounted in 2% agarose and then submitted to microscopy. Five different snapshots in time are shown. The original movies are part of the supplementary data.</p