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SAXS Analysis and Characterization of Anticancer Activity of PNP-UDP Family Protein from Putranjiva roxburghii

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Abstract

A class of plant defense and storage proteins, including Putranjiva roxburghii PNP protein (PRpnp), belongs to PNP-UDP family. The PRpnp and related plant proteins contain a disrupted PNP-UDP domain as revealed in previous studies. In PRpnp, the insert disrupting the domain contains the trypsin inhibitory site. In the present work, we analyzed native PRpnp (nPRpnp) complex formation with trypsin and inosine using SAXS experiments and established its dual functionality. Results indicated a relatively compact nPRpnp:Inosine structure, whereas trypsin complex showed conformational changes/flexibility. nPRpnp also exhibited a strong anti-cancer activity toward breast cancer (MCF-7), prostate cancer (DU-145) and hepatocellular carcinoma (HepG2) cell lines. MCF-7 and DU-145 were more sensitive to nPRpnp treatment as compared to HepG2. However, nPRpnp treatment showed no effect on the viability of HEK293 cells indicating that nPRpnp is specific for targeting the viability of only cancer cells. Further, acridine orange, DAPI and DNA fragmentation studies showed that cytotoxic effect of nPRpnp is mediated through induction of apoptosis as evident from the apoptosis-associated morphological changes and nuclear fragmentation observed after PRpnp treatment of cancer cells. These results suggest that PRpnp has the potential to be used as an anticancer agent. This is first report of anticancer activity as well as SAXS-based analysis for a PNP enzyme with trypsin inhibitory activity.

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Data Availability

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Code Availability

Not Applicable.

Abbreviations

nPRpnp:

Putranjiva roxburghii PNP protein purified from its native source

SAXS:

Small-angle X-ray scattering

MTT:

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide

AO/EB:

Acridine orange and ethidium bromide

DAPI:

4’,6-Diamidino-2-phenylindole

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Acknowledgements

PV and BK thank CSIR, SPSY thanks DST and RV thanks UGC for financial support. We also acknowledge Dr Ashish from the CSIR IMTECH for in-house SAXS facility for SAXS data collection.

Funding

The authors did not receive support from any organization for the submitted work.

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Authors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, 247 667, India

    Preeti Verma, Ritu Varshney, Bibekananda Kar, Partha Roy & Ashwani K. Sharma

  2. CSIR-Institute of Microbial Technology, Chandigarh, 160 036, India

    Shiv Pratap Singh Yadav

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  1. Preeti Verma
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  2. Ritu Varshney
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  3. Shiv Pratap Singh Yadav
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  4. Bibekananda Kar
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  5. Partha Roy
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  6. Ashwani K. Sharma
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Contributions

The study was conceived by AKS. PV, RV, SPSY and BK designed and performed all experiments. Cell line studies were guided by PR. PV prepared the first draft of the manuscript. SPSY, PR, and AKS revised the manuscript. All authors approved the final manuscript.

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Correspondence to Ashwani K. Sharma.

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Verma, P., Varshney, R., Yadav, S.P.S. et al. SAXS Analysis and Characterization of Anticancer Activity of PNP-UDP Family Protein from Putranjiva roxburghii. Protein J 41, 381–393 (2022). https://doi.org/10.1007/s10930-022-10060-x

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