Difference between revisions of "Part:BBa K5129001"

Revision as of 14:25, 1 October 2024

NSP4+PNC-27

The present composite part consists of the fused NSP4 signaling peptide for protein export to periplasmic space(BBa_K3606042) and PNC-27 anticancer peptide(BBa_K5129000). The NSP4 signaling peptide exports the PNC-27 to the periplasm, after which it is cleaved out by signal peptidases

Overview

Aiming to address the problem of complications presented due to conventional breast adenocarcinoma therapy methods, we are proposing an innovative solution - bacteriotherapy, using non-pathogenic chassis E.coli to synthesize anticancer peptide. Bacteria will serve the synthesis purpose, therefore cannot directly interact with cancer cells. That is why the E.coli will be enclosed in the hydrogel network. The specificity of the treatment is ensured by using lactate sensor and peptide specificity itself. In order to prevent spreading out of the bacteria inside the body, the kill switch was designed.

Anticancer peptide

PNC-27 is a 32-residue peptide composed of an HDM2 binding domain of p53 (residues 12–26) and CPP leader sequence. The peptide is synthetic in nature, meaning that it was initially produced through protein engineering methods.

Penetratin sequence

CPP leader sequence represents part essential for binding and entrance into target cells. The fragment is also known as Penetratin. It was essentially derived from a leader sequence of the antennapedia protein [1]. Penetratin contains a high density of positively charged residues that stabilize an α-helix when present on its carboxyl terminal end [1]. Because of this property, aside from the main function Penetratin is essential for proper folding of PNC-27.

HDM2 binding domain

PNC-27 has been shown to eradicate cancer cells with higher specificity due to the nature of its binding partner, indicating that normal cells are typically not affected by it [2,3]. Human Double Minute Homolog 2 or HDM-2, is known to be overexpressed in cancerous cells [3]. Through binding to HDM2, PNC-27 becomes cytotoxic for cancer cells as this interaction leads to the formation of pores on cell membranes [4]. Direct binding to HDM-2 is conducted via α-helical conformation of the protein [1]. HDM-2 is overexpressed in the membranes of both solid and non-solid tissue tumors [3]. The experimental results suggest that early developing tumor cells exhibit high concentrations of HDM-2 in their membranes [5,6]. In addition, HDM-2 was reported to be a marker of rapidly growing tumors. Its elevated levels correlate with metastatic properties of primary tumor cell cultures obtained from breast cancer patients [7]. Cancer cells obtain these motility properties due to co-localization of peptide with E-cadherin in the cancer cells plasma membranes, which leads to the ubiquitination and degradation of the latter.

Treatment efficiency

PNC-27 demonstrated its efficiency in a wide variety of cancer cell lines. For instance, PNC-27 induced rapid total cell necrosis (within 1 hr) of several breast cancer cell lines [8]. The results of another study show that PNC-27 is cytotoxic to cells from long-established and chemotherapy-resistant human ovarian cancer cell lines [9]. Necrosis of cells was confirmed as elevated concentrations of lactate dehydrogenase (LDH) were released from the samples treated with the peptide [3]. IC50 values of the peptide range from 75 ug/ml (18.6 uM) to 200 ug/ml (50 uM) [3]. Notably, the studies reported that PNC-27 induced pores in the membranes of cancer cells, but cell membrane lysis was not observed after treatment of untransformed cells [10, 7, 11]. Lastly, for the in vivo experiments, PNC-27 was tested on human pancreatic cancer cells (MIA-PaCa-2) and a melanoma cell line (A2058) in nude mice. While efficient tumor eradication was observed, no evidence of toxic side effects was documented [3]. The proposed mechanism of treatment is visualized in Figure 1. PNC-27 binds to HDM-2 creating complexes that coalesce to form transmembrane pores.

Signalling peptide

To reach the cancer cell membranes, the peptide must escape the bacteria cell. This can be accomplished by using signaling peptides, connected to the main peptide, PNC-27. Signaling peptides should guide the molecule to the membrane of E.coli and export it outside of the cell. For this project, we chose NSP4 as the signaling peptide and fused it to PNC-27. Essentially, unfolded precursors composed of the chimeric proteins get translocated across the cytoplasmic membrane of bacteria, which is followed by cleavage of the signal peptide by specific signal peptidase. As a result, the peptide gets folded into the native structure and gets exported from the periplasmic space of bacteria.

Regarding the structure, signaling peptides are composed of 3 structural domains, each having a distinct function [11]. The amino terminal part (n-region) has a positive charge. It’s typically followed by a hydrophobic h-region. The C-region contains a protein recognition sequence, which is the site through which the signaling peptide gets separated from the rest of the protein.

The following properties were considered for selection of the signaling peptide:

• Must be native for E. coli to ensure proper cleavage and release of the native PNC27.
• E. coli K-12 in the UniProtKB database indicates that the median signal sequence length in E. coli is 22 amino acids, with a minimum of 15–16 amino acids.

NSP4, derived from DsbAss native to E.coli, was chosen because it suits the above-described requirements and due to the following reasons [12]:

• Section of the peptide is mediated via the secretory (Sec) pathway.
  • The peptide gets recognized by signal recognition particles (SRP) via the fifty-four homolog (Ffh) region of SRP. Subsequently, SRP guides proteins for export into the periplasmic space [13, 14].
• Other works reported higher secretion efficiency compared to other similar sequences. For instance, NSP4 improved secretion of ATH35L by Escherichia coli by four times compared to conventional DsbAss signaling peptide [12]. Induction time was also reduced.
• Signaling peptidase performs very precise cleavage on the recognition site.

Below is the sequence of NSP4, with the recognition site for signaling peptidase highlighted in yellow: MKKITAAAGLLLLAAQPAMA

Sequence and Features