Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.

• MeSH
• energetický metabolismus MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• organely metabolismus   ultrastruktura   MeSH
• oxidace-redukce MeSH
• proteom metabolismus   MeSH
• proteomika MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• regulace genové exprese MeSH
• shluková analýza MeSH
• síra metabolismus   MeSH
• Trichomonas vaginalis genetika   metabolismus   MeSH
• železo metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH
• protozoální proteiny MeSH
• síra MeSH
• železo MeSH

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.

• MeSH
• anaerobióza MeSH
• genetické vektory genetika   MeSH
• Giardia lamblia cytologie   genetika   MeSH
• hydrolasy genetika   MeSH
• ligandy MeSH
• mitochondrie metabolismus   MeSH
• molekulární zobrazování metody   MeSH
• organely metabolismus   MeSH
• protozoální proteiny genetika   MeSH
• rekombinantní fúzní proteiny genetika   MeSH
• reportérové geny genetika   MeSH
• Trichomonas vaginalis cytologie   genetika   MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• ligandy MeSH
• protozoální proteiny MeSH
• rekombinantní fúzní proteiny MeSH

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.

• MeSH
• Giardia lamblia metabolismus   MeSH
• mitochondriální proteiny analýza   chemie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• paraziti metabolismus   MeSH
• proteom analýza   metabolismus   MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• shluková analýza MeSH
• tandemová hmotnostní spektrometrie MeSH
• velikost mitochondrií fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteom MeSH