Hybrid protein-peptide system for the selective pH-dependent binding and photodynamic ablation of cancer cells.

Creating new tools for the early diagnosis and treatment of cancer is one of the most important and intensively developing areas of modern medicine. Currently, photodynamic cancer therapy (PDT) is attracting increasing attention as a unique modality of minimally invasive treatment and due to the absence of acquired resistance. However, PDT is associated with undesirable activities, such as non-specific photodynamic effects of sunlight on healthy tissues. Therefore, an important fundamental task is the development of improved PDT agents that selectively act on the affected areas. Here, we report the development of a hybrid protein-peptide system for the selective pH-dependent binding and subsequent photodynamic cancer cells ablation. It is known that a distinctive feature of cancer cells is a decreased pH level in the extracellular space. In this study we exploited a peptide fragment (pHLIP) as a targeting module, which spontaneously binds and embeds into the cell membrane when pH decreases below neutral. A mutant of miniSOG protein fused to pHLIP was used as a photosensitizing constituent. We demonstrate that this protein-peptide photosensitizing system selectively binds to HeLa cells at pH below 6.8 and kills them when exposed to light. These findings demonstrate the feasibility of using genetically encoded MiniSOG fusions with pHLIP for the targeted delivery of PSs to cancer cells and subsequent highly precise photodynamic therapy.

Co-expression of different proteins in Escherichia coli using plasmids with identical origins of replication.

It is generally accepted that the use of two different plasmids with the identical origins of replication in bacteria is not desirable due to their "incompatibility". The utilization of the same bacterial enzymatic apparatus for replication of different plasmids is thought to cause a significant redistribution in favor of one of them. In the present work, examining co-expression of two different fluorescent proteins in Escherichia coli, we have shown that the use of highly homologous plasmids with identical origins of replication and providing resistance to different antibiotics results in high representation of both plasmids in bacteria. Meanwhile, the level of gene expression and the amount of proteins produced may differ and is determined mostly by their sequence rather than by the "incompatibility" of the plasmids.

Cancer cells targeting with genetically engineered constructs based on a pH-dependent membrane insertion peptide and fluorescent protein.

The targeted delivery of nanodrugs to malignant neoplasm is one of the most pressing challenges in the development of modern medicine. It was reported earlier that a bacteriorhodopsin-derived pH low insertion peptide (pHLIP) targets acidic tumors and has the ability to translocate low molecular weight cargoes across the cancer cell membrane. Here, to better understand the potential of pHLIP-related technologies, we used genetically engineered fluorescent protein (EGFP) as a model protein cargo and examined targeting efficiencies of EGFP-pHLIP hybrid constructs in vitro with the HeLa cell line at different pHs. By two independent monitoring methods we observed an increased binding affinity of EGFP-pHLIP fusions to HeLa cells at pH below 6.8. Confocal images of EGFP-pHLIP-treated cells showed bright fluorescence associated with the cell membrane and fluorescent dots localized inside the cell, that became brighter with time. To elucidate the pHLIP-mediated EGFP cell entry mechanisms, we performed a series of experiments with specific inhibitors of endocytosis. Our results imply that EGFP-pHLIP internalization is realized by endocytosis of various types.

Abnormal pigment epithelium-derived factor processing in progressive myopia.

Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of "soluble" 45 kDa PEDF reduced by 2-fold, while the content of "insoluble" 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.