The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.
- MeSH
- chromatin * MeSH
- dimerizace MeSH
- DNA metabolismus MeSH
- lidé MeSH
- mezibuněčné signální peptidy a proteiny * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Accurate estimates of intermolecular interaction energy, ΔE, are crucial for modeling the properties of organic electronic materials and many other systems. For a diverse set of 50 dimers comprising up to 50 atoms (Set50-50, with 7 of its members being models of single-stacking junctions), benchmark ΔE data were compiled. They were obtained by the focal-point strategy, which involves computations using the canonical variant of the coupled cluster theory with singles, doubles, and perturbative triples [CCSD(T)] performed while applying a large basis set, along with extrapolations of the respective energy components to the complete basis set (CBS) limit. The resulting ΔE data were used to gauge the performance for the Set50-50 of several density-functional theory (DFT)-based approaches, and of one of the localized variants of the CCSD(T) method. This evaluation revealed that (1) the proposed "silver standard" approach, which employs the localized CCSD(T) method and CBS extrapolations, can be expected to provide accuracy better than two kJ/mol for absolute values of ΔE, and (2) from among the DFT techniques, computationally by far the cheapest approach (termed "ωB97X-3c/vDZP" by its authors) performed remarkably well. These findings are directly applicable in cost-effective yet reliable searches of the potential energy surfaces of noncovalent complexes.
- MeSH
- benchmarking * MeSH
- dimerizace MeSH
- elektronika * MeSH
- fyzikální jevy MeSH
- polymery MeSH
- Publikační typ
- časopisecké články MeSH
Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti-inflammatory therapies.
CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
- MeSH
- Bacteria enzymologie genetika MeSH
- Bacteroidetes enzymologie genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Cas proteiny metabolismus MeSH
- CRISPR-Cas systémy * MeSH
- dimerizace MeSH
- DNA primery biosyntéza MeSH
- DNA-dependentní DNA-polymerasy genetika metabolismus MeSH
- DNA-primasa genetika metabolismus MeSH
- Escherichia coli metabolismus MeSH
- exprese genu MeSH
- fylogeneze MeSH
- mutace MeSH
- prokaryotické buňky metabolismus MeSH
- rekombinantní proteiny MeSH
- ribonukleotidy metabolismus MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.
- MeSH
- B-lymfocyty imunologie MeSH
- COVID-19 imunologie MeSH
- dimerizace MeSH
- epitopy imunologie MeSH
- genové produkty env - virus lidské imunodeficience chemie genetika imunologie MeSH
- glykoprotein S, koronavirus imunologie MeSH
- glykosylace MeSH
- HIV infekce imunologie MeSH
- HIV protilátky imunologie MeSH
- HIV-1 imunologie MeSH
- imunoglobuliny - Fab fragmenty chemie imunologie MeSH
- lidé MeSH
- Macaca mulatta MeSH
- neutralizující protilátky imunologie MeSH
- polysacharidy chemie imunologie MeSH
- receptory antigenů B-buněk chemie MeSH
- SARS-CoV-2 imunologie MeSH
- široce neutralizující protilátky imunologie MeSH
- vakcíny imunologie MeSH
- virus opičí imunodeficience genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The authors report on the synthesis and biological evaluation of new compounds whose structure combines tacrine and indole moieties. Tacrine-indole heterodimers were designed to inhibit cholinesterases and β-amyloid formation, and to cross the blood-brain barrier. The most potent new acetylcholinesterase inhibitors were compounds 3c and 4d (IC50 = 25 and 39 nM, respectively). Compound 3c displayed considerably higher selectivity for acetylcholinesterase relative to human plasma butyrylcholinesterase in comparison to compound 4d (selectivity index: IC50 [butyrylcholinesterase]/IC50 [acetylcholinesterase] = 3 and 0.6, respectively). Furthermore, compound 3c inhibited β-amyloid-dependent amyloid nucleation in the yeast-based prion nucleation assay and displayed no dsDNA destabilizing interactions with DNA. Compounds 3c and 4d displayed a high probability of crossing the blood-brain barrier. The results support the potential of 3c for future development as a dual-acting therapeutic agent in the prevention and/or treatment of Alzheimer's disease.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc farmakoterapie MeSH
- amyloidní beta-protein metabolismus MeSH
- cholinesterasové inhibitory chemie farmakologie MeSH
- cílená molekulární terapie MeSH
- dimerizace MeSH
- DNA chemie MeSH
- hematoencefalická bariéra MeSH
- indoly chemie farmakologie MeSH
- inhibiční koncentrace 50 MeSH
- lidé MeSH
- ligandy MeSH
- neuroprotektivní látky chemie farmakologie MeSH
- preklinické hodnocení léčiv MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- takrin chemie farmakologie MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.
- MeSH
- biokatalýza MeSH
- biologické modely MeSH
- dimerizace MeSH
- konformace proteinů MeSH
- messenger RNA genetika metabolismus ultrastruktura MeSH
- podjednotky ribozomu metabolismus MeSH
- proteiny z Escherichia coli chemie metabolismus ultrastruktura MeSH
- ribozomy metabolismus ultrastruktura MeSH
- stabilita RNA MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations in hcis-PT cause severe human diseases. Here, we reveal that hcis-PT exhibits a heterotetrameric assembly in solution, consisting of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and inactive Nogo-B receptor (NgBR) heterodimers. Importantly, the 2.3 Å crystal structure reveals that the tetramer assembles via the DHDDS C-termini as a dimer-of-heterodimers. Moreover, the distal C-terminus of NgBR transverses across the interface with DHDDS, directly participating in active-site formation and the functional coupling between the subunits. Finally, we explored the functional consequences of disease mutations clustered around the active-site, and in combination with molecular dynamics simulations, we propose a mechanism for hcis-PT dysfunction in retinitis pigmentosa. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.
- MeSH
- alkyltransferasy a aryltransferasy chemie genetika metabolismus MeSH
- aminokyselinové motivy MeSH
- dimerizace MeSH
- katalytická doména MeSH
- lidé MeSH
- mutace MeSH
- receptory buněčného povrchu chemie genetika metabolismus MeSH
- retinopathia pigmentosa enzymologie genetika MeSH
- transferasy chemie genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The first review article on steroid dimers by Li and Dias in 1997, followed by the second review and a book on steroid dimers by Nahar and Sarker in 2007 and 2012, respectively, covered steroid dimers reported until the end of 2010. Since then, there have been considerable amounts of research carried out on steroid dimers, prompting the need for another comprehensive review on this topic. Therefore, this present review appraises the literature published during the period 2011-2019 on various aspects of steroid dimers, including isolation from natural sources, synthesis and applications. A structured and systematic literature search was performed, using the key words: steroid dimer, steroidal dimer, dimeric steroid, bis-steroid, bis-steroidal conjugates, molecular umbrella, cephalostatins, ritterazines and crellastatins. Several databases like Web of Knowledge, Science Direct, PubMed and Google Scholar were consulted. During the period covered in this review, well over 200 new synthetic steroidal dimers, ring A-ring A connection being the major group, have been reported, only one natural steroid dimer has been isolated, and potential applications of steroid dimers in the treatment of cancers and tumors, and microbial infections have been indicated.
We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.
- MeSH
- barva MeSH
- cholesterol chemie MeSH
- dimerizace MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie MeSH
- fúze membrán * MeSH
- konfokální mikroskopie MeSH
- lipidy chemie MeSH
- lipopeptidy chemie MeSH
- peptidy chemie MeSH
- polysorbáty chemie MeSH
- rezonanční přenos fluorescenční energie MeSH
- unilamelární lipozómy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
