Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

Weber, Janine; Bao, Han; Hartlmüller, Christoph; Wang, Zhiqin; Windhager, Almut; Janowski, Robert; Madl, Tobias; Jin, Peng; Niessing, Dierk

doi:10.7554/elife.11297

Cited by 41 publications

(167 citation statements)

References 53 publications

(88 reference statements)

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“…16,20 Insights into the specific molecular mechanism by which Purα and Purβ repress Acta2 have been provided by studies that have uncovered the unique structural features of each protein. 25 This result is consistent with other biochemical data indicating that repeat III also corresponds to the dimerization domain in Purβ. 22,23 The solved x-ray crystal structures of the truncated repeat I-II region of Drosophila melanogaster (Dm) Purα and fulllength Borrelia burgdorferi (Bb) Purα showed that certain β-strand and α-helix forming sequences located in each repeat associate to form a so-called PUR domain.…”

Section: Introductionsupporting

confidence: 93%

“…Limited tryptic digestion of recombinant Mm Purβ revealed that the residues 29 to 302 are necessary for high affinity binding of the protein to a purine-rich ssDNA sequence from the Acta2 promoter. 25 With this new structural information in hand, we generated revised homology models of the Mm Purβ intermolecular domain and the intact Purβ homodimer. Homology modeling suggested that Mm Purβ repeats I and II together fold to form an intramolecular PUR domain while two repeat IIIs associate to form an intermolecular PUR domain.…”

Section: Discussionmentioning

confidence: 99%

“…22,23 The solved x-ray crystal structures of the truncated repeat I-II region of Drosophila melanogaster (Dm) Purα and fulllength Borrelia burgdorferi (Bb) Purα showed that certain β-strand and α-helix forming sequences located in each repeat associate to form a so-called PUR domain. 23,25 This distinction along with other structural differences may explain why Purβ functions as a dominant repressor of Acta2 in comparison to Purα when evaluated using both gain-of-function and loss-offunction approaches. 22,24 The more recently reported crystal structure of repeat III from Dm Purα confirmed that another PUR domain is formed by the intermolecular association of two repeat III sequences in a manner which mediates Purα dimerization.…”

Section: Introductionmentioning

confidence: 99%

“…Horseradish peroxidase (HRP)-conjugates of goat anti-rat IgG, goat anti-rabbit IgG, goat anti-mouse IgG, and mouse IgG κ-binding protein were procured from Santa Cruz Biotechnology, Inc., Dallas, TX.Updated computational homology models of the Mus musculus (Mm) Purβ monomer and homodimer were generated based on Dm Purα crystal structures: 3K44 (Purα repeats I-II, no DNA) and 5FGO (two Purα repeats III, no DNA) 24,25. Cell culture media were procured from Mediatech, Inc., Manassas, VA. Fetal bovine serum (FBS) was purchased from Sigma-Aldrich, St. Louis, MO.…”

mentioning

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: Introductionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

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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“…Sense foci also colocalize with Purine-rich element binding protein-alpha (Purα), Ran GTPase activating protein 1 (RanGAP1) and Adenosine deaminase B2 (ADARB2) [100,107,108]. Purα may recognize partially denatured RNA in a similar way as MBNL1 [83,109]. ADARB2 may participate in foci formation or maintenance, and its sequestration leads to hypersensitivity to excitotoxicity [100].…”

Section: Rna Toxicity and Foci In C9orf72-als/ftdmentioning

confidence: 99%

RNA toxicity and foci formation in microsatellite expansion diseases

Zhang

Ashizawa

2017

Current Opinion in Genetics & Development

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More than 30 incurable neurological and neuromuscular diseases are caused by simple microsatellite expansions consisted of 3–6 nucleotides. These repeats can occur in non-coding regions and often result in a dominantly inherited disease phenotype that is characteristic of a toxic RNA gain-of-function. The expanded RNA adopts unusual secondary structures, sequesters various RNA binding proteins to form insoluble nuclear foci, and causes cellular defects at a multisystem level. Nuclear foci are dynamic in size, shape and colocalization of RNA binding proteins in different expansion diseases and tissue types. This review sets to provide new insights into the disease mechanisms of RNA toxicity and foci modulation, in light of recent advancement on bi-directional transcription, antisense RNA, repeat-associated non-ATG translation and beyond.

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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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Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

Cited by 41 publications

References 53 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

RNA toxicity and foci formation in microsatellite expansion diseases

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

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