Single-Stranded DNA-Binding Proteins: Multiple Domains for Multiple Functions

Dickey, Thayne H.; Altschuler, Sarah E.; Wuttke, Deborah S.

doi:10.1016/j.str.2013.05.013

Cited by 106 publications

(130 citation statements)

References 128 publications

(169 reference statements)

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“…These actions seem to be caused by the inhibitory effect of DF on the interactions of tumor cells and nonmalignant cell compartments of the microenvironment, including stromal and ECs. Potential mechanisms that remain to be explored in the interaction between DF and different cell types could be the role of transporters that translocate nucleosides, single-stranded DNA-binding proteins, 43 and charge dependent interactions as those reported for heparin. 44 In conclusion, the present study provides the first evidence that DF interacts with the cell membrane of ECs of different origin and becomes internalized.…”

Section: Discussionmentioning

confidence: 99%

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

Palomo

Mir

Rovira

et al. 2016

Blood

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Key Points• Specific interaction of DF with EC membranes is followed by its internalization mainly through macropinocytic mechanisms.• DF attachment to the cell membrane is sufficient to perform its antiinflammatory and antioxidant effects on the endothelium.Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization.Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations. (Blood. 2016;127(13):1719-1727 Introduction Defibrotide (DF) is a mixture of 90% single-stranded phosphodiester oligonucleotides (length, 9-80 mer; average molecular mass, 16.5 6 2.5 KDa) and 10% double-stranded phosphodiester oligonucleotides, derived from the controlled depolymerization of porcine intestinal mucosal DNA.1-3 Several functions, specially related to hemostasis, have been ascribed to DF. 4 In this regard, our group has demonstrated the protective effect of DF on the endothelium, by preventing the endothelial damage associated with hematopoietic cell transplantation (HCT) conditions, 5,6 and with the deleterious effect of immunosuppresants. 7 In our in vitro endothelial activation model, DF has exhibited reproducible effects on endothelial cells (ECs) from different origins. DF demonstrates antiinflammatory, antithrombotic, and antiapoptotic properties. However, although its effects are increasingly better understood, its precise mechanism of action remains to be elucidated.There is limited knowledge about DF pharmacokinetics, pharmacodynamics, and mechanisms of action. [8][9][10] However, 2 distinct properties of ...

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Section: Discussionmentioning

confidence: 99%

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

Palomo

Mir

Rovira

et al. 2016

Blood

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“…In contrast to bacterial SSBs, eukaryotic SSBs generally function as a heterotrimer consisting of six OB folds distributed among three subunits; however, only four of these OB folds are involved in binding to ssDNA (7), which results in an extended arrangement for the protein-ssDNA complex. Telomere end binding proteins, which safeguard the vulnerable telomere 3′ end of ssDNA, also use different numbers of OB folds to bind with ssDNA (8). These proteins show high sequence specificities for their respective telomeric sequences, and this sequence preference may be linked to their function as telomeres (8)(9)(10).…”

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confidence: 99%

“…Telomere end binding proteins, which safeguard the vulnerable telomere 3′ end of ssDNA, also use different numbers of OB folds to bind with ssDNA (8). These proteins show high sequence specificities for their respective telomeric sequences, and this sequence preference may be linked to their function as telomeres (8)(9)(10). Furthermore, very few SSBs have been structurally characterized as using the K homology domain (three α-helices packed against a three-stranded β-sheet) and RNA recognition motif domains (8) to form a complex with ssDNA.…”

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confidence: 99%

Molecular determinants of the interactions between proteins and ssDNA

Mishra

Levy

2015

Proc. Natl. Acad. Sci. U.S.A.

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ssDNA binding proteins (SSBs) protect ssDNA from chemical and enzymatic assault that can derail DNA processing machinery. Complexes between SSBs and ssDNA are often highly stable, but predicting their structures is challenging, mostly because of the inherent flexibility of ssDNA and the geometric and energetic complexity of the interfaces that it forms. Here, we report a newly developed coarse-grained model to predict the structure of SSBssDNA complexes. The model is successfully applied to predict the binding modes of six SSBs with ssDNA strands of lengths of 6-65 nt. In addition to charge-charge interactions (which are often central to governing protein interactions with nucleic acids by means of electrostatic complementarity), an essential energetic term to predict SSB-ssDNA complexes is the interactions between aromatic residues and DNA bases. For some systems, flexibility is required from not only the ssDNA but also, the SSB to allow it to undergo conformational changes and the penetration of the ssDNA into its binding pocket. The association mechanisms can be quite varied, and in several cases, they involve the ssDNA sliding along the protein surface. The binding mechanism suggests that coarse-grained models are appropriate to study the motion of SSBs along ssDNA, which is expected to be central to the function carried out by the SSBs.ssDNA | protein-DNA interaction | coarse-grained model

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“…Besides, some specific ssDNA-processing proteins are recruited and coordinated by ssDNA binding proteins during DNA metabolism pathways (33)(34)(35). In spite of high sequence, structural and functional divergence, almost all classical ssDNA binding proteins contain one of the following four structural topologies: oligonucleotide/oligosaccharide/oligopeptide-binding (OB) folds, K homology (KH) domains, RNA recognition motifs (RRMs), and whirly domains (36).…”

Section: Discussionmentioning

confidence: 99%