The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment.

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The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2297-2308.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2297-2308 ◽

Cited By ~ 60

Author(s):

Daniel Menendez ◽

Alberto Inga ◽

Michael A. Resnick

Keyword(s):

Dna Binding ◽

Target Genes ◽

Tumor Development ◽

Human Tumor ◽

Human Cells ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

The Relationship ◽

Biological Outcomes

ABSTRACT Human tumor suppressor p53 is a sequence-specific master regulatory transcription factor that targets response elements (REs) in many genes. p53 missense mutations in the DNA-binding domain are often cancer associated. As shown with systems based on the yeast Saccharomyces cerevisiae, p53 mutants can alter the spectra and intensities of transactivation from individual REs. We address directly in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA-binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV or ionizing radiation, or spontaneously. These patterns reflected changes in the spectra and activities of target genes, as demonstrated for P21, MDM2, BAX, and MSH2. Thus, as originally proposed for “master genes of diversity,” p53 mutations in human cells can differentially influence target gene transactivation, resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.

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Substitutions at Nonconserved Rheostat Positions Modulate Function by Rewiring Long-Range, Dynamic Interactions

Molecular Biology and Evolution ◽

10.1093/molbev/msaa202 ◽

2020 ◽

Vol 38 (1) ◽

pp. 201-214 ◽

Cited By ~ 1

Author(s):

Paul Campitelli ◽

Liskin Swint-Kruse ◽

S Banu Ozkan

Keyword(s):

Dna Binding ◽

Long Range ◽

Binding Domain ◽

Dna Binding Domain ◽

Dynamic Coupling ◽

Long Distance ◽

Repressor Protein ◽

Functional Changes ◽

Lactose Repressor ◽

Wide Range

Abstract Amino acid substitutions at nonconserved protein positions can have noncanonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein. This region contains nonconserved positions for which substitutions alter DNA-binding affinity. We first chose to study 11 substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA-binding positions, and the degree of flexibility change correlated with experimentally measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA-binding positions in a manner that captured other experimentally determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA-binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated hardly any substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type lactose repressor protein, the dynamic couplings between the DNA-binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and noncanonical substitutions outcomes at nonconserved positions arise from rewiring long-range communication among functionally important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

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Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile

Blood ◽

10.1182/blood-2013-05-501072 ◽

2013 ◽

Vol 122 (7) ◽

pp. 1293-1304 ◽

Cited By ~ 82

Author(s):

Kimberly Cramer-Morales ◽

Margaret Nieborowska-Skorska ◽

Kara Scheibner ◽

Michelle Padget ◽

David A. Irvine ◽

...

Keyword(s):

Dna Binding ◽

Gene Mutation ◽

Expression Profile ◽

Synthetic Lethality ◽

Binding Domain ◽

Dna Binding Domain ◽

Key Points ◽

Peptide Aptamer ◽

Domain I

Key Points Targeting RAD52 DNA binding domain I by peptide aptamer induces synthetic lethality in BRCA-deficient leukemias. Individual patients with BRCA-deficient leukemias could be identified by genetic and epigenetic profiling.

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Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain

ACS Nano ◽

10.1021/acsnano.6b05938 ◽

2016 ◽

Vol 11 (1) ◽

pp. 144-152 ◽

Cited By ~ 23

Author(s):

Armando Hernandez-Garcia ◽

Nicole A. Estrich ◽

Marc W. T. Werten ◽

Johan R. C. Van Der Maarel ◽

Thomas H. LaBean ◽

...

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain ◽

Dna Templates ◽

Protein Polymer ◽

Wide Range

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Substitutions at Non-Conserved Rheostat Positions Modulate Function by Re-Wiring Long-Range, Dynamic Interactions

10.1101/2020.07.02.185207 ◽

2020 ◽

Cited By ~ 1

Author(s):

Paul Campitelli ◽

Liskin Swint-Kruse ◽

S. Banu Ozkan

Keyword(s):

Dna Binding ◽

Long Range ◽

Protein Function ◽

Binding Domain ◽

Dna Binding Domain ◽

Dynamic Coupling ◽

Long Distance ◽

Modulate Function ◽

Functional Changes ◽

Wide Range

AbstractAmino acid substitutions at nonconserved protein positions can have non-canonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities (“DFI”) and dynamic coupling (“DCI”) for positions in the linker region of the lactose repressor protein (“LacI”). This region contains nonconserved positions for which substitutions alter DNA binding affinity. We first chose to study eleven substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA binding positions, and the degree of flexibility change correlated with experimentally-measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA binding positions in a manner that captured other experimentally-determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated almost no substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type LacI, the dynamic couplings between the DNA binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and non-canonical substitutions outcomes at nonconserved positions arise from re-wiring long-range communication among functionally-important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

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A Quantitative Systems Approach to Define Novel Effects of Tumour p53 Mutations on Binding Oncoprotein MDM2

International Journal of Molecular Sciences ◽

10.3390/ijms23010053 ◽

2021 ◽

Vol 23 (1) ◽

pp. 53

Author(s):

Manuel Fuentes ◽

Sanjeeva Srivastava ◽

Angela M. Gronenborn ◽

Joshua LaBaer

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Hot Spot ◽

Point Mutations ◽

Protein Microarrays ◽

Binding Domain ◽

Structural Domain ◽

Dna Binding Domain ◽

Label Free ◽

Wide Range

Understanding transient protein interactions biochemically at the proteome scale remains a long-standing challenge. Current tools developed to study protein interactions in high-throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self-assembling protein microarrays produced by freshly translating full-length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label-free platform that measures many protein interactions in real-time allowing the determination of the KDs and rate constants. The developed novel NAPPA-SPRi technique showed excellent ability to study protein-protein interactions of clinical mutants of p53 with its regulator MDM2. Furthermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin-3, currently in clinical practice, which usually disrupts the p53-MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg-273-Cys, Arg-273-His, Arg-248-Glu, Arg-280-Lys), on the structural domain (His-179-Tyr, Cys-176-Phe), on hydrophobic moieties in the DNA binding domain (Arg-280-Thr, Pro-151-Ser, Cys-176-Phe) and hot spot mutants (Gly-245-Cys, Arg-273-Leu, Arg-248-Glu, Arg-248-Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.

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