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Biochemical and subcellular characterization of a squid hnRNPA/B-like protein 2 in osmotic stress activated cells reflects molecular properties conserved in this protein family

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Abstract

Background

We have identified endogenous p65 to be an SDS-stable dimer protein composed of ~ 37 kDa hnRNPA/B-like subunits. We have investigated molecular properties involved in the stability of dimeric form, and their regulation in the transition between monomeric and dimeric forms of hnRNPA/B-like protein 2. We also investigated a cellular property conserved between squid hnRNPA/B-like protein 2 and human hnRNPA1 protein in a neuronal context.

Methods and results

Here we show biochemical properties of a recombinant hnRNPA/B-like protein 2 (rP2) in vitro experiments, as one of p65 subunit. We found that interaction between rP2 and RNA molecules interfered with the dynamics of rP2 dimers formation, involved in disulfide bonds and/or postranslational alterations in distinct stage of SDS-stable dimers formation. In addition, we have performed immunofluorescence in SH-SY5Y cells and observed that the pEGFP-P2 fusion protein was expressed in the nucleus, similar to what is observed for human hnRNPA1 protein.

Conclusion

Our results reinforce the idea that p65 is an SDS-stable dimer. Thus, a deeper understanding between monomeric and dimeric transition dynamic is critical into evolution of several neurodegenerative disease.

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Data availability

All data generated in this study are available in the manuscript. This manuscript is published in the bioRxi as a preprint https://doi.org/10.1101/2021.07.02.450876

Abbreviations

rP2:

Recombinant squid hnRNPA/B-like protein 2

pEGFP-P2:

PEGFP-C1 vector containing the ORF of squid hnRNPA/B-like protein 2

mCherry-P2:

PmCherry-C1 vectors containing the ORF of squid hnRNPA/B-like protein 2

PABP-1:

Poly-A binding protein-1

DND1:

Dead end protein homolog1

hnRNP:

Heterogeneous nuclear ribonucleoprotein

ORF:

Open reading frame

PAGE:

Polyacrylamide gel electrophoresis

PBS:

Phosphate buffered saline

RNP:

Ribonucleoprotein

RNP1/RNP2:

Core sequences of RNA recognition motifs

SDS:

Sodium dodecyl sulfate

SG:

Stress granules

BFS:

Bovine fetal serum

FRAP:

Fluorescence recovery after photobleach

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Acknowledgements

Thanks to Silvia Andrade and Domingos Pitta for expert technical help, and M.Sc Elizabete R. Milani for technical help with confocal microscopy, performed at Laboratório Multiusuário de Microscopia Confocal—LMMC, Fapesp 2004/08868-0. Special thanks to Lexie Friend, PhD, Honorary Fellow School of Chemistry and Molecular Biosciences at the University of Queensland St Lucia, 4072, Australia. She did make suggestions important to the manuscript and my works that it supported my finds in discussion section

Funding

The Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); no specifc grant was received for this Publisher.

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Authors and Affiliations

  1. Department Cellular & Molecular Biology, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, 14049-900, Brazil

    Gabriel S. Lopes & Diego T. P. Lico

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  1. Gabriel S. Lopes
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  2. Diego T. P. Lico
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Contributions

DTPL (performed experiments biochemical and prepared manuscript); GSL (performed experiments immunofluorescence); MLPL (analyzed data) and REL (designed the experiments). and Contributions Structural the Marine Resources Center of the Marine Biological Laboratory in Woods Hole, MA and f the Centro de Biologia Marinha-CEBIMar, University of São Paulo, São Sebastião, Brazil.

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Correspondence to Diego T. P. Lico.

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The authors declare that the research was conducted in the absence of conflict of interest.

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The number of animals used has been conformed to ethical guidelines presented by National System of Nature Conservation Units Snuc-Ibama/Brazil with solicitation Nº 53615US and Public Health Service, National Institutes of Health Publication according to Law No. 9,985/2000.

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Lopes, G.S., Lico, D.T.P. Biochemical and subcellular characterization of a squid hnRNPA/B-like protein 2 in osmotic stress activated cells reflects molecular properties conserved in this protein family. Mol Biol Rep 49, 4257–4268 (2022). https://doi.org/10.1007/s11033-022-07260-0

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