Combining Unique Multiplex Gateway Cloning and Bimolecular Fluorescence Complementation (BiFC) for High-Throughput Screening of Protein–Protein Interactions

Grbeša

et al. 2018

Sci Rep

Self Cite

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

“…Proteins were separated on 6–12% sodium dodecyl sulfate-polyacrylamide resolving gels. Immunoblotting was performed as described previously 50 using the antibodies listed in Table 2 .…”

Section: Methodsmentioning

confidence: 99%

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest

Grbeša

et al. 2018

Sci Rep

Self Cite

Journal of Biological Chemistry

“…To verify this newly identified SOD2 domain in hsp70-mediated binding, we used bimolecular fluorescence complementation (BiFC) analysis to directly visualize SOD2/hsp70 interactions in live cells (22)(23)(24). Hsp70 constructs were made with deletion of amino acid residues 393-537 to express recombinant hsp70 mutant (hsp70⌬393-537) protein that could not FIGURE 1.…”

Section: Domain Mapping Of the Sod2 And Hsp70mentioning

confidence: 99%

Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function

Afolayan

Alexander

Holme

et al. 2017

Edited by Linda SpremulliStress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at 445 GERAMT 450 . Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo. To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu 446 and Arg 447 cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.

“…Many groups have explored the interactions of transcription factors with other proteins as well as with DNA using different versions of PCAs [97][98][99][100][101]. Recently the generation of large collections of vectors that allow performing large-scale PPI detection both in vitro and in living organisms has further increased the value of such techniques [102,103].…”

Section: Protein-fragment Complementation Assay (Pca)mentioning

confidence: 99%

Biophysical Techniques for Target Validation and Drug Discovery in Transcription-Targeted Therapy

Moustaqil

Gambin

Sierecki

2020

IJMS

In the post-genome era, pathologies become associated with specific gene expression profiles and defined molecular lesions can be identified. The traditional therapeutic strategy is to block the identified aberrant biochemical activity. However, an attractive alternative could aim at antagonizing key transcriptional events underlying the pathogenesis, thereby blocking the consequences of a disorder, irrespective of the original biochemical nature. This approach, called transcription therapy, is now rendered possible by major advances in biophysical technologies. In the last two decades, techniques have evolved to become key components of drug discovery platforms, within pharmaceutical companies as well as academic laboratories. This review outlines the current biophysical strategies for transcription manipulation and provides examples of successful applications. It also provides insights into the future development of biophysical methods in drug discovery and personalized medicine.