Part:BBa_K5129001

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.

Figure 1. Proposed model for pore formation based on PNC-27-HDM-2 complexes.

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 red: MKKITAAAGLLLLAAQPAMA

Modelling

Introduction

General introduction

In science, mathematical models are necessary for formulating hypotheses, making predictions, understanding complex systems, estimating parameters, optimizing processes, and interpreting data. Because they pose no risk, scientists can learn about potentially dangerous situations. When studies provide sample information, they aid in data analysis and preliminary data interpretation to support scientists in conclusions. Additionally, these models serve as a scientific language, facilitating discussions and knowledge advancement among a range of specialists.

Structural bioinformatics analysis for PNC-27 and NSP4-PNC-27

Figure 2. Summary of BLAST protein-protein alignment of PNC-27 to p53.

A)

B)

C)

Figure 3. Color coded PyMOL visualization of PNC-27 structure.

As PNC-27 is known to be a chimeric protein composed of two parts, the p53 HDM-2 binding domain, and cell-penetrating sequence, it was beneficial to understand which peptide fragments correspond to these parts. To do so, we obtained an amino acid sequence and PDB ID (1Q2I) of PNC-27 from UniProt. Subsequently, amino PNC-27 was analyzed through the BLAST tool by aligning the amino acid sequences of PNC-27, p53, and the cell-penetrating peptide (CPP) leader of antennapedia protein. For p53, the HDM-2 binding domain, which corresponds to residues 12−26, was utilized. The summary is illustrated on Figure 2. After alignment, the structure was visualized via PyMOL, and each of the components was color-coded on the protein: yellow corresponds to the p53 fragment, and cyan corresponds to the CPP sequence. Figure 3 demonstrates the obtained results: A - cartoon representation, B - mesh representation, C - surface representation.

A)

B)

C)

Figure 3. Color coded PyMOL visualization of chimeric peptides’ (NSP4+PNC-27) structure.

Subsequently, as NSP4 was fused to PNC-27 on the amino-terminus, the newly obtained sequence had to be visualized via PyMOL as well. Firstly, we needed to obtain a PDB file of the structure. The SWISS-MODEL tool was used for structure prediction. However, as the tool performs modeling based on sequence homology alignment, the NSP4 component of the chimeric protein did not appear on the obtained structures. The most plausible explanation for this error is the absence of a template covering the region, meaning that the sequence similarity of proteins in the corresponding library is very low. Hence, the NSP4 part was omitted in the model. To overcome this challenge, the peptide analysis was conducted using the AlphaFold server, which was designed for protein 3D model prediction from submitted amino acid sequences. The software predicted chimeric peptides’ structure, which was then downloaded as a PDB file and visualized through PyMOL, the results of which can be accessed in Figure 3 (A - cartoon representation, B - mesh representation, C - surface representation). Yellow color corresponds to the p53 fragment, and cyan corresponds to the CPP sequence, and orange fragment corresponds to NSP4 signaling peptide. As can be seen by the images, addition of signaling peptide drastically changes the conformation of the chimeric protein, meaning that functioning of PNC-27 might be hindered by these modifications. Hence, antitumor activity can be completely lost. Therefore, it was highly beneficial to choose the signaling peptide, which gets cleaved from the native protein at specific sites with high accuracy and efficiency, which is the case for NSP4. Essentially, NSP4 will be removed by the corresponding signaling peptidase native to E.coli after translocation of the chimeric protein to periplasmic space through recognition by SRP and subsequent transport via SEC pathway. Afterwards, PNC-27 folds into its native structure, as described by structural bioinformatics moedling, which can then perform normal functions of the protein.

A)

B)

Figure 4. Color coded PyMOL visualization of PNC-27 docking to HDM-2.

After visualizing the structure of PNC-27 and its chimeric counterpart, protein-protein docking indicating the binding of PNC-27 to its target, HDM-2, was performed by submitting PDB files of both proteins to the ClusPro 2.0 tool. Subsequently, several potential models for this binding were obtained, each described by their respective coefficient weight values:

Only balanced coefficients were read. Cluster 0, with a Center Weighted Score of -1174.3 and Lowest Energy of -1212.7, was chosen for further analysis. Lastly, visualization with PyMOL was repeated as described before, and the results can be accessed through Figure 4 (A- cartoon representation, B - surface representation).

As for the construction of the plasmid, the genetic sequence of PNC-27 was not available on open sources prior to our work. Therefore, the sequence had to be computed for the creation of the PNC-27 coding part. To do so, we utilized amino acid alignments of the peptide to p53 and CPP obtained for the visualization. Afterward, appropriate amino acid fragments of both components of PNC-27 were aligned to the mRNA sequences that code for these amino acids in native proteins from which the synthetic construct was made. BLAST alignment was used for this analysis as well. Then, reverse DNA sequences coding these mRNA fragments were manually computed, which was followed by codon optimization and insertion of the obtained parts into the plasmid designed for this project through SnapGene. Hence, we were able to compute a new part coding PNC-27 anticancer peptide having only its amino acid sequence.

References

[1] Kanovsky, M., Raffo, A., Drew, L., Rosal, R., Do, T., Friedman, F. K., Rubinstein, P., Visser, J., Robinson, R., Brandt-Rauf, P. W., Michl, J., Fine, R. L., & Pincus, M. R. (2001b). Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells. Proceedings of the National Academy of Sciences, 98(22), 12438–12443. https://doi.org/10.1073/pnas.211280698

[2] Sarafraz-Yazdi, E., Mumin, S., Cheung, D., Fridman, D., Lin, B., Wong, L., Rosal, R., Rudolph, R., Frenkel, M., Thadi, A., Morano, W. F., Bowne, W. B., Pincus, M. R., & Michl, J. (2022). PNC-27, a Chimeric p53-Penetratin Peptide Binds to HDM-2 in a p53 Peptide-like Structure, Induces Selective Membrane-Pore Formation and Leads to Cancer Cell Lysis. Biomedicines, 10(5), 945. https://doi.org/10.3390/biomedicines10050945

[3] Thadi, A., Gleeson, E. M., Khalili, M., Shaikh, M. F., Goldstein, E., Morano, W. F., Daniels, L. M., Grandhi, N., Glatthorn, H., Richard, S. D., Campbell, P. M., Sarafraz-Yazdi, E., Pincus, M. R., & Bowne, W. B. (2020, September 1). Anti-Cancer tumor cell necrosis of epithelial ovarian cancer cell lines depends on high expression of HDM-2 protein in their membranes. http://www.annclinlabsci.org/content/50/5/611.full

[4] Oncolyze. (n.d.). Oncolyze. https://www.oncolyze.com/science

[5] Shaikh, M. F., Morano, W. F., Lee, J., Gleeson, E., Babcock, B. D., Michl, J., Sarafraz-Yazdi, E., Pincus, M. R., & Bowne, W. B. (2016, December 1). Emerging role of MDM2 as target for Anti-Cancer therapy: A review. http://www.annclinlabsci.org/content/46/6/627

[6] Sarafraz-Yazdi, E., Bowne, W. B., Adler, V., Sookraj, K. A., Wu, V., Shteyler, V., Patel, H., Oxbury, W., Brandt-Rauf, P., Zenilman, M. E., Michl, J., & Pincus, M. R. (2010). Anticancer peptide PNC-27 adopts an HDM-2-binding conformation and kills cancer cells by binding to HDM-2 in their membranes. Proceedings of the National Academy of Sciences, 107(5), 1918–1923. https://doi.org/10.1073/pnas.0909364107

[7] Michl, J., Scharf, B., Schmidt, A., Huynh, C., Hannan, R., Von Gizycki, H., Friedman, F. K., Brandt‐Rauf, P., Fine, R. L., & Pincus, M. R. (2006). PNC‐28, a p53‐derived peptide that is cytotoxic to cancer cells, blocks pancreatic cancer cell growth in vivo. International Journal of Cancer, 119(7), 1577–1585. https://doi.org/10.1002/ijc.22029

[8] Ex vivo Efficacy of Anti-Cancer Drug PNC-27 in the Treatment of Patient-Derived Epithelial Ovarian Cancer. (n.d.). PubMed. https://pubmed.ncbi.nlm.nih.gov/26663795/

[9] Sarafraz-Yazdi E, Mumin S, Cheung D, Fridman D, Lin B, Wong L, Rosal R, Rudolph R, Frenkel M, Thadi A, Morano WF, Bowne WB, Pincus MR, Michl J. PNC-27, a Chimeric p53-Penetratin Peptide Binds to HDM-2 in a p53 Peptide-like Structure, Induces Selective Membrane-Pore Formation and Leads to Cancer Cell Lysis. Biomedicines. 2022 Apr 20;10(5):945. doi: 10.3390/biomedicines10050945. PMID: 35625682; PMCID: PMC9138867.

[10] Sarafraz-Yazdi E., Gorelick C., Wagreich A.R., Salame G., Angert M., Gartman C.H., Gupta V., Bowne W.B., Lee Y.C., Abulafia O., et al. Ex vivo Efficacy of Anti-Cancer Drug PNC-27 in the Treatment of Patient-Derived Epithelial Ovarian Cancer. Ann. Clin. Lab. Sci. 2015;45:650–658.

[11] Owji, Hajar & Nezafat, Navid & Negahdaripour, Manica & HajiEbrahimi, Ali & Younes, Ghasemi. (2018). A Comprehensive Review of Signal Peptides: Structure, Roles, and Applications. European Journal of Cell Biology. 97. 10.1016/j.ejcb.2018.06.003.

[12] Han S, Machhi S, Berge M, Xi G, Linke T, Schoner R. Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express. 2017 Dec;7(1):93. doi: 10.1186/s13568-017-0394-1. Epub 2017 May 12. PMID: 28497288; PMCID: PMC5427057.

[13] Soares, C. R., Gomide, F. I., Ueda, E. K., & Bartolini, P. (2003). Periplasmic expression of human growth hormone via plasmid vectors containing the PL promoter: use of HPLC for product quantification. Protein Engineering Design and Selection, 16(12), 1131–1138. https://doi.org/10.1093/protein/gzg114

[14] Luirink, J., & Dobberstein, B. (1994). Mammalian and Escherichia coli signal recognition particles. Molecular Microbiology, 11(1), 9–13. https://doi.org/10.1111/j.1365-2958.1994.tb00284.x

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 48
  
• 1000
  COMPATIBLE WITH RFC[1000]