Novel Hybrid Catalysts of Cysteine Proteases Enhanced by Chitosan and Carboxymethyl Chitosan Micro- and Nanoparticles.

Micro- and nanoparticles of chitosan and carboxymethyl chitosan were synthesized, both with and without ascorbic acid. Methods were developed to form complexes between these micro- and nanoparticles and plant proteases-ficin, papain, and bromelain. It was demonstrated that the activity of cysteine protease complexes with carboxymethyl chitosan micro- and nanoparticles was higher compared to those with chitosan micro- and nanoparticles.

Complexation of Bromelain, Ficin, and Papain with the Graft Copolymer of Carboxymethyl Cellulose Sodium Salt and -Vinylimidazole Enhances Enzyme Proteolytic Activity.

This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with -vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier.

Identification of acquired Notch3 dependency in metastatic Head and Neck Cancer.

During cancer development, tumor cells acquire changes that enable them to invade surrounding tissues and seed metastasis at distant sites. These changes contribute to the aggressiveness of metastatic cancer and interfere with success of therapy. Our comprehensive analysis of "matched" pairs of HNSCC lines derived from primary tumors and corresponding metastatic sites identified several components of Notch3 signaling that are differentially expressed and/or altered in metastatic lines and confer a dependency on this pathway.

Carboxymethyl Cellulose-Based Polymers as Promising Matrices for Ficin Immobilization.

The present work is devoted to research on the interaction between carboxymethyl cellulose sodium salt and its derivatives (graft copolymer of carboxymethyl cellulose sodium salt and ,-dimethyl aminoethyl methacrylate) with cysteine protease (ficin). The interaction was studied by FTIR and by flexible molecular docking, which have shown the conjugates' formation with both matrices. The proteolytic activity assay performed with azocasein demonstrated that the specific activities of all immobilized ficin samples are higher in comparison with those of the native enzyme.

Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features.

Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, -(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and -(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively).

Thermodynamics of globular protein native structure.

The temperature dependence of the partial heat capacity of the native protein structure in an aqueous solution has been analyzed. It is shown that the strictly linear temperature dependence is due to the contributions of the vibrational and conformational components, which indicates volume consistensy and the absence of conformational transitions up to the main two-state transition. The two-level structural and functional organization of the protein three-dimensional structure are considered in relation to the energy and conformational entropy properties in accordance with the principles of the organization of the protein macromolecule.

Novel biotechnological formulations of cysteine proteases, immobilized on chitosan. Structure, stability and activity.

Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively).

Light-induced effects in glycine aqueous solution studied by Fourier transform infrared-emission spectroscopy and ultraviolet-visible spectroscopy.

Although amino acids are insensitive to visible light, as is generally accepted, we show that particular light-matter interaction can break this obviousness. Using sensitive (FT-IR)-technique in a combination of a broadband visible light source, we registered emission spectra of glycine in the range 2500-500 cm. Sensitivity of the infrared emission spectrum to the exciting power -induced changes in the glycine structure was demonstrated experimentally.

Influence of UV radiation on molecular structure and catalytic activity of free and immobilized bromelain, ficin and papain.

Our research has shown that the degree of photosensitivity of the cysteine proteases can be arranged in the following order: bromelain → ficin → papain. After the UV irradiation with 151 J·m intensity of a bromelain solution, the enzyme activity has increased. No decrease in the catalytic capacity and the change in the size of the molecule was recorded in the 151-6040 J·m range of irradiation intensities.

Immobilization of inulinase on KU-2 ion-exchange resin matrix.

We develop a technique for the sorption of inulinase from Kluyveromyces marxianus on the KU-2 matrix cation-exchanger. The most appropriate conditions for immobilization are: 25 °C, pH 4.5, incubation time 1.

Evofosfamide for the treatment of human papillomavirus-negative head and neck squamous cell carcinoma.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line-derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models.

Thermodynamics of globular proteins.

The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the 'gas phase'. It was shown that specific values of the energy of weak intramolecular bonds (∆H), conformational free energy (∆G) and entropy (∆S) are the same for proteins with molecular weight 7-25 kDa.

In silico design of high-affinity ligands for the immobilization of inulinase.

Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase - an enzyme that cleaves inulin and fructose-containing polymers to fructose - has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking. The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine).

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields.

Interaction of phospholipase A of the E. coli outer membrane with the inhibitors of eucaryotic phospholipases A₂ and their effect on the Ca²⁺-induced permeabilization of the bacterial membrane.

Phospholipase A of the bacterial outer membrane (OMPLA) is a β-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A₂--palmitoyl trifluoromethyl ketone (PACOCF₃) and aristolochic acid (AA)--with OMPLA and considers a possible involvement of the enzyme in the Ca²⁺-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF₃ and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine.

Structural and dynamic properties of thymopoietin mimetics.

We propose a hypothesis that the T-cell receptor is a possible target of thymic hormones. We modelled the conformational dynamics of thymopentin and its structural variants in solution, as well as the interactions of these short peptides with the proposed molecular target. Thymopentin is a five-amino-acid fragment of the thymic hormone thymopoietin (residues 32 to 36) that reproduces the immunomodulatory activity of the complete hormone.

The entropic nature of protein thermal stabilization.

We performed thermodynamic analysis of temperature-induced unfolding of mesophilic and thermophilic proteins. It was shown that the variability in protein thermostability associated with pH-dependent unfolding or linked to the substitution of amino acid residues on the protein surface is evidence of the governing role of the entropy factor. Numerical values of conformational components in enthalpy, entropy and free energy which characterize protein unfolding in the "gas phase" were obtained.

Small molecule antagonists of the Wnt/β-catenin signaling pathway target breast tumor-initiating cells in a Her2/Neu mouse model of breast cancer.

Background: Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure.

Methods/findings: To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro.

Constant serum levels of secreted asialoglycoprotein receptor sH2a and decrease with cirrhosis.

Aim: To investigate the existence and levels of sH2a, a soluble secreted form of the asialoglycoprotein receptor in human serum.

Methods: Production of recombinant sH2a and development of a monoclonal antibody and an enzyme-linked immunosorbent assay (ELISA). This assay was used to determine the presence and concentration of sH2a in human sera of individuals of both sexes and a wide range of ages.

A secreted form of the asialoglycoprotein receptor, sH2a, as a novel potential noninvasive marker for liver fibrosis.

Background And Aim: The human asialoglycoprotein receptor is a membrane heterooligomer expressed exclusively in hepatocytes. A soluble secreted form, sH2a, arises, not by shedding at the cell surface, but by intracellular cleavage of its membrane-bound precursor, which is encoded by an alternatively spliced form of the receptor H2 subunit. Here we determined and report that sH2a, present at constant levels in serum from healthy individuals is altered upon liver fibrosis, reflecting the status of hepatocyte function.

Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer.

Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures.

PERK-dependent compartmentalization of ERAD and unfolded protein response machineries during ER stress.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the ER membrane kinases PERK and IRE1 leading to the unfolded protein response (UPR). We show here that UPR activation triggers PERK and IRE1 segregation from BiP and their sorting with misfolded proteins to the ER-derived quality control compartment (ERQC), a pericentriolar compartment that we had identified previously. PERK phosphorylates translation factor eIF2alpha, which then accumulates on the cytosolic side of the ERQC.