Polyprotein synthesis: a journey from the traditional pre-translational method to modern post-translational approaches for single-molecule force spectroscopy.

Polyproteins, an array of protein units of similar or differential functions in tandem, have been extensively utilized by organisms, unicellular or multicellular, as concentrators of the myriad of molecular activities. Most eukaryotic proteins, two-thirds in unicellular organisms, and more than 80% in metazoans, are polyproteins. Although the use of polyproteins continues to evolve in nature, our understanding of the structure-function-property of polyproteins is still limited. Cumbersome recombinant strategies and the lack of convenient in vitro synthetic routes of polyproteins have been rate-determining factors in the progress. However, in this review we have discussed the revolutionary journey of polyprotein synthesis with a major focus on surface-based structure-function-property studies, especially using force spectroscopy at the single-molecule level.

Transient interactions drive the lateral clustering of cadherin-23 on membrane.

Cis and trans-interactions among cadherins secure multicellularity. While the molecular structure of trans-interactions of cadherins is well understood, work to identify the molecular cues that spread the cis-interactions two-dimensionally is still ongoing. Here, we report that transient, weak, yet multivalent, and spatially distributed hydrophobic interactions that are involved in liquid-liquid phase separations of biomolecules in solution, alone can drive the lateral-clustering of cadherin-23 on a membrane. No specific cis-dimer interactions are required for the lateral clustering. In cells, the cis-clustering accelerates cell-cell adhesion and, thus, contributes to cell-adhesion kinetics along with strengthening the junction. Although the physiological connection of cis-clustering with rapid adhesion is yet to be explored, we speculate that the over-expression of cadherin-23 in M2-macrophages may facilitate faster attachments to circulatory tumor cells during metastasis.

Instantaneous splicing and excision of inteins to synthesize polyproteins on a substrate with tunable linkers.

Nature has adapted chimeric polyproteins to achieve superior and multiplexed functionality in a single protein. However, the hurdles in in vitro synthesis have restricted the biomimicry of and subsequent fundamental studies on the structure-function relationship of polyproteins. Recombinant expression of polyproteins and the synthesis of polyproteins via the enzyme-mediated repetitive digestion and ligation of individual protein domains have been widely practiced. However, recombinant expression often suffers from an in vitro refolding process, whereas enzyme-assisted peptide conjugation results in heterogeneous products, primarily due to enzymatic re-digestion, and prolonged and multistep reactions. Moreover, both methods incorporate enzyme-recognition residues of varying lengths as artifacts at interdomain linkers. The linkers, although tiny, regulate the spatiotemporal conformations of the polyproteins differentially and tune the folding dynamics, stability, and functions of the constituent protein. In an attempt to leave no string behind at the interdomain junctions, here, we develop a 'splice and excise' synthetic route for polyproteins on a substrate using two orthogonal split inteins. Inteins self-excise and conjugate the protein units covalently and instantaneously, without any cofactors, and incorporate a single cysteine or serine residue at the interdomain junctions.

Development and characterization of pellets for targeted delivery of 5-fluorouracil and phytic acid for treatment of colon cancer in Wistar rat.

The present study was designed to investigate the therapeutic efficacy of metal chelator and anticancer drug in the treatment of colorectal cancer (CRC). Pellets containing Phytic acid, 5- Fluorouracil (5-FU), Microcrystalline cellulose (MCC) PH 101, Hydroxypropyl Methylcellulose (HPMC) and Barium sulfate were prepared by using extrusion spheronization technique. Prepared pellets were coated with Eudragit S100 to achieve colon-specific drug delivery. Pellets were characterized for various pharmaceutical and micromeritic attributes. The in vivo therapeutic efficacy comprising of both pharmacokinetic and pharmacodynamic parameters was determined in Ehrlich ascites carcinoma (EAC) induced cancer animal model. Phytic acid and 5-FU combinations seem to exert higher cytotoxic activity via increased reactive oxygen species (ROS) level by chelating manganese. Further pharmacokinetic studies reveled approximately 50% lower Cmax in the finished formulation, indicates lower systemic exposure to the drug. X-ray radiography ensures the localized delivery of the encapsulated drug. Histopathological studies indicated no significant local toxicity compared to the uncoated formulation. Results inferred that the proposed combination has superior anticancer activity with minimum systemic and local toxicity and it opens a new avenue in the treatment of colorectal cancer.

The critical role of bisphosphonates to target bone cancer metastasis: an overview.

Cancer becomes the leading cause of deaths worldwide, including breast cancer, prostate cancer and lung cancer that preferentially metastasize to bone and bone marrow. Bisphosphonates (BPs) have been used successfully for many years to reduce the skeletal complications related with the benign and malignant bone diseases that are characterized by enhanced osteoclastic bone resorption. Nitrogen-containing bisphosphonates (N-BPs) have also been demonstrated to exhibit direct anti-tumour effects. BPs binds avidly to the bone matrix, and released from matrix during bone resorption process, BPs are internalized by the osteoclasts where they interfere with biochemical pathways and induce osteoclast apoptosis. BPs also antagonizes the production of osteoclast and promotes the osteoblasts proliferation. Currently, Zoledronic acid is widely used as one of the BP having high bone specificity and potential to inhibit the osteoclast-mediated bone resorption. In addition to inhibition of cell multiplication and initiation of apoptosis in cultured cancer cells, they also interfere with adhesion of cancer cells to the bone matrix and inhibit cell migration and invasion. Pathophysiology and current target therapies like conjugate of BPs with liposomes, nanoparticle used for the treatment of bone cancer is reviewed in this article along with the use of different BPs.