Heat shock protein 70kDa (HSP70) is a highly conserved protein family expressed by most organisms and found in both prokaryotes and eukaryotes. All the members of the HSP70 protein family have a molecular weight that ranges around 70kDa and all share the same chaperone function (Radons, 2016). They bind to short hydrophobic polypeptide sequences in their substrate binding domain (SBD) with an affinity that is allosterically modulated by the N-terminal adenine nucleotide binding domain (NBD) (Feige and Polla, 1994). When adenosine triphosphate is bound to the NBD the affinity for the substrate in the SBD is low but when adenosine diphosphate is bound, the affinity increases (Clerico et al., 2015). This process actually triggers the closing of the C-terminal α-helical domain. This domain acts as a lid that retains the substrate bound to the rigid structure of the SBD (Strub et al., 2003). HSP70 has two major roles in the cells; first, it binds to peptidic parts of nascent proteins and keeps them from premature folding (Bukau et al., 2000, Hartl and Hayer-Hartl, 2002); second, HSP70 has a protective feature in response to intense cellular stress such as environmental, physiological or pathological stress (Qu et al., 2015, Pinto et al., 1991). HSP70 is of major importance in the regulation and protection of intracellular proteins. Indeed, HSP70, besides being closely involved in the folding of newly produced proteins as a chaperone protein, is also involved in many other intracellular regulative pathways. When a substrate binds to HSP70, several pathways can be triggered (Kim et al., 2018). The substrate can be refolded, either directly by the action of HSP70 or through the recruitment of co-chaperones (Sharma and Masison, 2009), but can also be translocated to autophagosome or proteasome for intracellular recycling (Witkin et al., 2017). HSP70 can also trigger regulations of transcription factors that lead to an indirect regulation of the proteome (Pratt and Toft, 2003). Under an intense stress, HSP70 is able to bind to already formed proteins and to protect them against denaturation and aggregation (Rokutan, 2000). All these possible pathways triggered by HSP70 give this protein a major role in maintaining cell functions. HSP70 can also play an important role as a therapeutic agent in neurodege- nerative diseases caused by peptide aggregations such as α-synuclein in Parkinson’s disease (Rochet et al., 2012) or amyloid-β and Tau in Alzheimer’s disease (Campanella et al., 2018).
Modulating intracellular levels of HSP70 has the potential to be an efficient therapeutic action (Kim et al., 2018) and more particularly an acute increase of its concentration could give a temporary protection against protein aggregation and misfolding. There are two methods to increase the level of a specific protein in cells. Either transfecting an oligonucleotide or a plasmid DNA coding for this protein (Kim and Eberwine, 2010) or delivering directly the wanted protein inside the cell (Pisal et al., 2010). The transfection of oligonucleotides and plasmid DNA can be either permanent if the sequence is integrated in the genome or transient if not. In either case, the expression of the protein relies on the cell machinery and is dependent on transcription factor. If a protein is delivered instead, it is directly available for a biological effect the delivery will then be transient as the cells will not express the protein after its natural degradation. Delivering a protein is an alternative to oligonucleotide and plasmid DNA delivery and presents advantages for acute and non-permanent treatment. It also allows a more straight-forward treatment strategy as it does not rely on the cell transcription machinery (Lee et al., 2019).
Cell-penetrating peptides (CPPs) are a class of peptides able to translocate through biological barriers and to transport bioactive macromolecules directly inside cells (Bechara and Sagan, 2013). PepFect14 is an amphipathic CPP composed by a 21 amino acids sequence and a stearic acid tail bound to its N-terminus (Ezzat et al., 2011). It is able to form non covalent complexes with its cargoes via electrostatic interactions (Lehto et al., 2017). It has been shown for several years already that PepFect14 formed complexes with diverse modified or non-modified oligonucleotides – siRNA (Ervin et al., 2019), plasmid DNA (Veiman et al., 2013) and antisense oligonucleotides (Ezzat et al., 2011) - and mediated their transfection inside various types of cells. The complexes, when placed in cell culture media, formed nanoparticle with a hydrodynamic diameter in the range of 102 nm (Lehto et al., 2017). It was shown in a previous study from our group that the formation of nanoparticles was a key factor in the ability of PepFect14 to mediates transfections (Lehto et al., 2016). In another study where we studied the gene regulations induced by the translocation of PepFect14 in HeLa cells, we demonstrated that HSPA1B, a gene that codes for a HSP70 protein, was up-regulated as a reaction to the uptake of PepFect14. In the same study, we showed that a binding opportunity between PepFect14 and HSP70 existed in the polypeptide binding site (Dowaidar et al., 2017).
In the present study, we assessed the ability of PepFect14 to form a complex with HSP70. We also characterized the particles that these complexes formed in cell culture media before monitoring the uptake of a fluorescent-labelled HSP70 in cells and finally we detected a biological effect induced by the transfected HSP70.