Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and <i>Acta2</i> Repression by Purine-Rich Element-Binding Protein B

Rumora, Amy E.; Ferris, Lauren A.; Wheeler, Tamar R.; Kelm, Robert J.

doi:10.1021/acs.biochem.6b00006

Cited by 6 publications

(36 citation statements)

References 48 publications

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“…23,40 Hence, a P223L substitution in the linker region may alter the spatial orientation of intermolecular and intramolecular domains in a manner which enhances MSY1-binding affinity. In contrast to results of comparative DNA-binding assays, quantitative analysis of the binding of Purβ to corepressor partner MSY1 indicated that the P223L and R297Q substitutions altered protein-protein interaction.…”

Section: Discussionmentioning

confidence: 99%

“…40 Some modifications to the process were necessary to optimize the production and purity of the Purβ variants as outlined in the Supporting Information. 40 Some modifications to the process were necessary to optimize the production and purity of the Purβ variants as outlined in the Supporting Information.…”

Section: Recombinant Protein Purificationmentioning

confidence: 99%

“…40 Briefly, each protein was diluted to concentrations ranging from 75 to 300 μg/mL in buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0 with 10 mM β-mercaptoethanol, and 5× SYPRO Orange. 40 Briefly, each protein was diluted to concentrations ranging from 75 to 300 μg/mL in buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0 with 10 mM β-mercaptoethanol, and 5× SYPRO Orange.…”

Section: Thermal Shift Assaymentioning

confidence: 99%

“…40 The compositions of the coating, blocking, binding, and wash buffers were the same as those used in the solid-phase protein-DNA interaction assay except where noted. 40 The compositions of the coating, blocking, binding, and wash buffers were the same as those used in the solid-phase protein-DNA interaction assay except where noted.…”

Section: Quantitative Purβ-msy1 Interaction Assaymentioning

confidence: 99%

“…40 Briefly, purified Purβ WT or variant proteins at 100 nM were immobilized on the wells of EIA/RIA plates in coating buffer composed of 20 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl 2 -6H 2 O, 0.5 mM DTT, and 5.0 μg/mL BSA for 17 hours at 4°C. 40 Briefly, purified Purβ WT or variant proteins at 100 nM were immobilized on the wells of EIA/RIA plates in coating buffer composed of 20 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl 2 -6H 2 O, 0.5 mM DTT, and 5.0 μg/mL BSA for 17 hours at 4°C.…”

Section: Purβ Antibody Screening Assaymentioning

confidence: 99%

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Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

Ferris

Kelm

2018

J of Cellular Biochemistry

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Purine‐rich element‐binding protein B (Purβ) inhibits myofibroblast differentiation by repressing the expression of the smooth muscle α‐actin gene (Acta2). Several reports have identified the structural domains in Purβ that enable its characteristic interaction with purine‐rich single‐stranded DNA (ssDNA) sequences in the Acta2 promoter. However, little is known about the physical and functional effects of single‐nucleotide polymorphisms that alter individual amino acid residues in Purβ. This study evaluated seven rare single amino acid variants of human PURB engineered into the homologous mouse Purβ protein. Mapping the location of variant residues on a homology model of the Purβ homodimer suggested that most of the altered residues are remote from the predicted ssDNA‐binding regions of the protein. The repressor activity of each Purβ variant was assessed in transfected fibroblasts and smooth muscle cells via Acta2 promoter‐reporter assays. A Q64* nonsense variant was completely inactive while missense variants exhibited repressor activity that ranged from ~1.5‐fold greater to ~2‐fold less than wild‐type Purβ. Lower activity variants P223L and R297Q were expressed in bacteria and purified to homogeneity. Each variant was physically indistinguishable from wild‐type Purβ in terms of quaternary structure and thermostability. Results of DNA and protein‐binding assays indicated that the P223L and R297Q variants retained high affinity and specificity for purine‐rich ssDNA sequences but differed in their interaction with other Acta2 regulatory proteins. These findings suggest that the presence of certain variant residues affects the Acta2 repressor activity of Purβ by altering its interaction with other transcription factors but not with ssDNA.

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Section: Discussionmentioning

confidence: 99%

Section: Recombinant Protein Purificationmentioning

confidence: 99%

Section: Thermal Shift Assaymentioning

confidence: 99%

Section: Quantitative Purβ-msy1 Interaction Assaymentioning

confidence: 99%

Section: Purβ Antibody Screening Assaymentioning

confidence: 99%

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Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

Ferris

Kelm

2018

J of Cellular Biochemistry

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Characterization of purine‐rich element binding protein B as a novel biomarker in acute myelogenous leukemia prognostication

Kelm

Lamba

Levis

et al. 2017

J of Cellular Biochemistry

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Acute myelogenous leukemia (AML) is an aggressive hematologic cancer characterized by infiltration of proliferative, clonal, abnormally differentiated cells of myeloid lineage in the bone marrow and blood. Malignant cells in AML often exhibit chromosomal and other genetic or epigenetic abnormalities that are useful in prognostic risk assessment. In this study, the relative expression and novel single-stranded DNA (ssDNA) binding function of purine-rich element binding proteins A and B (Purα and Purβ) were systematically evaluated in established leukemia cell lines and in lineage committed myeloid cells isolated from patients diagnosed with a hematologic malignancy. Western blotting revealed that Purα and Purβ are markedly elevated in CD33 /CD66b cells from AML patients compared to healthy subjects and to patients with other types of myeloid cell disorders. Results of in silico database analysis of PURA and PURB mRNA expression during hematopoiesis in conjunction with the quantitative immunoassay of the ssDNA-binding activities of Purα and Purβ in transformed leukocyte cell lines pointed to Purβ as the more distinguishing biomarker of myeloid cell differentiation status. Purβ ssDNA-binding activity was significantly increased in myeloid cells from AML patients but not from individuals with other myeloid-related diseases. The highest levels of Purβ activity were detected in myeloid cells from primary AML patients and from AML patients displaying other risk factors forecasting a poor prognosis. Collectively, these findings suggest that the enhanced ssDNA-binding activity of Purβ in transformed myeloid cells may serve as a unique and measurable phenotypic trait for improving prognostic risk stratification in AML.

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Computational investigation of proton transfer, pKa shifts and pH-optimum of protein-DNA and protein-RNA complexes

Peng

Alexov

2017

Proteins

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Protein-nucleic acid interactions play a crucial role in many biological processes. This work investigates the changes of pKa values and protonation states of ionizable groups (including nucleic acid bases) that may occur at protein-nucleic acid binding. Taking advantage of the recently developed pKa calculation tool DelphiPka, we utilize the large protein-nucleic acid interaction database (NPIDB database) to model pKa shifts caused by binding. It has been found that the protein's interfacial basic residues experience favorable electrostatic interactions while the protein acidic residues undergo proton uptake to reduce the energy cost upon the binding. This is in contrast with observations made for protein-protein complexes. In terms of DNA/RNA, both base groups and phosphate groups of nucleotides are found to participate in binding. Some DNA/RNA bases undergo pKa shifts at complex formation, with the binding process tending to suppress charged states of nucleic acid bases. In addition, a weak correlation is found between the pH-optimum of protein-DNA/RNA binding free energy and the pH-optimum of protein folding free energy. Overall, the pH-dependence of protein-nucleic acid binding is not predicted to be as significant as that of protein-protein association. Proteins 2017; 85:282-295. © 2016 Wiley Periodicals, Inc.

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Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element-Binding Protein B

Cited by 6 publications

References 48 publications

Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

Characterization of purine‐rich element binding protein B as a novel biomarker in acute myelogenous leukemia prognostication

Computational investigation of proton transfer, pKa shifts and pH-optimum of protein-DNA and protein-RNA complexes

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