Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• ferredoxiny metabolismus   MeSH
• jaderné lokalizační signály MeSH
• lyasy štěpící vazby C-S chemie   genetika   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• proteiny asociované s jadernou matrix chemie   genetika   metabolismus   MeSH
• proteiny vázající železo metabolismus   MeSH
• protozoální proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• Trypanosoma brucei brucei enzymologie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Intrinsically disordered regions (IDRs) are protein regions that lack persistent secondary or tertiary structure under native conditions. IDRs represent >40% of the eukaryotic proteome and play a crucial role in protein-protein interactions. The classical approach for identification of these interaction interfaces is based on mutagenesis combined with biochemical techniques such as coimmunoprecipitation or yeast two-hybrid screening. This approach either provides information of low resolution (large deletions) or very laboriously tries to precisely define the binding epitope via single amino acid substitutions. Here, we report the use of a peptide microarray based on the human scaffold protein AXIN1 for high-throughput and -resolution mapping of binding sites for several AXIN1 interaction partners in vitro For each of the AXIN1-binding partners tested, i.e. casein kinase 1 ϵ (CK1ϵ); c-Myc; peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1); and p53, we found at least three different epitopes, predominantly in the central IDR of AXIN1. We functionally validated the specific AXIN1-CK1ϵ interaction identified here with epitope-mimicking peptides and with AXIN1 variants having deletions of short binding epitopes. On the basis of these results, we propose a model in which AXIN1 competes with dishevelled (DVL) for CK1ϵ and regulates CK1ϵ-induced phosphorylation of DVL and activation of Wnt/β-catenin signaling.

• MeSH
• axin protein metabolismus   MeSH
• beta-katenin metabolismus   MeSH
• čipová analýza proteinů metody   MeSH
• fosforylace MeSH
• interakční proteinové domény a motivy * MeSH
• kaseinkinasa Iepsilon metabolismus   MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• peptidy metabolismus   MeSH
• protein dishevelled metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• signální dráha Wnt MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• albuminy metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• sekreční proteiny prostaty chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

DNA repair scaffolds mediate specific DNA and protein interactions in order to assist repair enzymes in recognizing and removing damaged sequences. Many scaffold proteins are dedicated to repairing a particular type of lesion. Here, we show that the budding yeast Saw1 scaffold is more versatile. It helps cells cope with base lesions and protein-DNA adducts through its known function of recruiting the Rad1-Rad10 nuclease to DNA. In addition, it promotes UV survival via a mechanism mediated by its sumoylation. Saw1 sumoylation favors its interaction with another nuclease Slx1-Slx4, and this SUMO-mediated role is genetically separable from two main UV lesion repair processes. These effects of Saw1 and its sumoylation suggest that Saw1 is a multifunctional scaffold that can facilitate diverse types of DNA repair through its modification and nuclease interactions.

• MeSH
• analýza přežití MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• oprava DNA * MeSH
• poškození DNA * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   genetika   metabolismus   MeSH
• sumoylace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

• MeSH
• databáze proteinů MeSH
• interleukin-10 metabolismus   MeSH
• konformace proteinů MeSH
• počítačová simulace MeSH
• proteinové inženýrství MeSH
• proteiny chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• ribozomy metabolismus   MeSH
• řízená evoluce molekul metody   MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this work, we report on the preparation of a novel biodegradable textile scaffold made of palmitoyl-hyaluronan (palHA). Monofilament fibres of palHA with a diameter of 120μm were prepared by wet spinning. The wet-spun fibres were subsequently processed into a warp-knitted textile. To find a compromise between swelling in water and degradability of the final textile scaffold, a series of palHA derivatives with different degrees of substitution of the palmitoyl chain was synthesized. Freeze-drying not only provided shape fixation, but also speeded up scaffold degradation in vitro. Fibronectin, fibrinogen, laminin and collagen IV were physically adsorbed on the textile surface to enhance cell adhesion on the material. The highest amount of adsorbed cell-adhesive proteins was achieved with fibronectin (89%), followed by fibrinogen (81%). Finally, textiles modified with fibronectin or fibrinogen both supported the adhesion and proliferation of normal human fibroblasts in vitro, proving to be a useful cellular scaffold for tissue engineering.

• MeSH
• biokompatibilní materiály chemie   metabolismus   farmakologie   MeSH
• buněčná adheze účinky léků   MeSH
• fibroblasty cytologie   účinky léků   MeSH
• hydrofobní a hydrofilní interakce * MeSH
• kyselina hyaluronová chemie   metabolismus   farmakologie   MeSH
• lidé MeSH
• povrchové vlastnosti MeSH
• textilie * MeSH
• tkáňové inženýrství MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

The human gene that encodes XRCC1 was cloned nearly thirty years ago but experimental analysis of this fascinating protein is still unveiling new insights into the DNA damage response. XRCC1 is a molecular scaffold protein that interacts with multiple enzymatic components of DNA single-strand break repair (SSBR) including DNA kinase, DNA phosphatase, DNA polymerase, DNA deadenylase, and DNA ligase activities that collectively are capable of accelerating the repair of a broad range of DNA single-strand breaks (SSBs). Arguably the most exciting aspect of XRCC1 function that has emerged in the last few years is its intimate relationship with PARP1 activity and critical role in preventing hereditary neurodegenerative disease. Here, I provide an update on our current understanding of XRCC1, and on the impact of hereditary mutations in this protein and its protein partners on human disease.

• MeSH
• DNA metabolismus   MeSH
• dvouřetězcové zlomy DNA MeSH
• jednořetězcové zlomy DNA MeSH
• lidé MeSH
• oprava DNA * MeSH
• protein XRCC1 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The 14-3-3 proteins, a family of conserved regulatory molecules, participate in a wide range of cellular processes through binding interactions with hundreds of structurally and functionally diverse proteins. Several distinct mechanisms of the 14-3-3 protein function were described, including conformational modulation of the bound protein, masking of its sequence-specific or structural features, and scaffolding that facilitates interaction between two simultaneously bound proteins. Details of these functional modes, especially from the structural point of view, still remain mostly elusive. This review gives an overview of the current knowledge concerning the structure of 14-3-3 proteins and their complexes as well as the insights it provides into the mechanisms of their functions. We discuss structural basis of target recognition by 14-3-3 proteins, common structural features of their complexes and known mechanisms of 14-3-3 protein-dependent regulations.

• MeSH
• DNA vazebné proteiny MeSH
• Eukaryota metabolismus   MeSH
• fosforylace MeSH
• genetická variace MeSH
• interakční proteinové domény a motivy MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• protein - isoformy chemie   metabolismus   MeSH
• proteiny 14-3-3 chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Bone and cartilage are tissues of a three-dimensional (3D) nature. Therefore, scaffolds for their regeneration should support cell infiltration and growth in all 3 dimensions. To fulfill such a requirement, the materials should possess large, open pores. Centrifugal spinning is a simple method for producing 3D fibrous scaffolds with large and interconnected pores. However, the process of bone regeneration is rather complex and requires additional stimulation by active molecules. In the current study, we introduced a simple composite scaffold based on platelet adhesion to poly-ε-caprolactone 3D fibers. Platelets were used as a natural source of growth factors and cytokines active in the tissue repair process. By immobilization in the fibrous scaffolds, their bioavailability was prolonged. The biological evaluation of the proposed system in the MG-63 model showed improved metabolic activity, proliferation and alkaline phosphatase activity in comparison to nonfunctionalized fibrous scaffold. In addition, the response of cells was dose dependent with improved biocompatibility with increasing platelet concentration. The results demonstrated the suitability of the system for bone tissue.

• MeSH
• adhezivita trombocytů účinky léků   MeSH
• alkalická fosfatasa metabolismus   MeSH
• kinetika MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• mezibuněčné signální peptidy a proteiny aplikace a dávkování   farmakologie   MeSH
• nádorové buněčné linie MeSH
• osteoblasty cytologie   účinky léků   ultrastruktura   MeSH
• osteogeneze účinky léků   MeSH
• polyestery chemie   farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• trombocyty účinky léků   metabolismus   ultrastruktura   MeSH
• tvar buňky účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH