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Electronic coupling to electrodes, Γ, as well as that across the examined molecules, H, is critical for solid-state electron transport (ETp) across proteins. Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron-mediating protein cytochrome c (CytC), measured in a monolayer configuration. Currents via CytC are temperature-independent between 30 and ~130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein-electrode binding significantly increases G, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy, Ea, of the ETp by more than a factor of two. The importance of H was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode-protein(-heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower Ea values (from high-temperature data) and higher conductance at low temperatures (in the temperature-independent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width). [ABSTRACT FROM AUTHOR]

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+ tumor cell lines. The inhibition of the antiproliferative effect of IFN-β by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-β to ErbB2-associated tumor cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] We have cloned genes coding for lipase and specific chaperone from Psychrobacter cryohalolentis. [•] We have purified and refolded the lipase in the presence of truncated chaperone. [•] Lip2Pc is a cold-adapted lipase active at wide temperature range from +5 to +65°C. [•] It is relatively stable above 60°C and in the presence of some metals and solvents. [•] Efficient refolding of lipase from P. cryohalolentis. [ABSTRACT FROM AUTHOR]

Enteropeptidase (synonym: enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide with kinetic parameters Km=0.16 mM and kcat=115 s−1 and small ester Z-Lys-SBzl with Km=140 μM, kcat=133 s−1. L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with Ki*=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions. [Copyright &y& Elsevier]

Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was -4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 μM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.

TRAIL is considered a promising antitumor agent because it causes apoptosis of transformed cells without affecting normal cells. However, many types of tumors are cytokine resistant, and combination therapy with various chemotherapeutic drugs is being developed to overcome the resistance. We have demonstrated that the combination of TRAIL with doxorubicin, bortezomib, and panobinostat dramatically reduced the viability of TRAIL-resistant A549 and HT-29 cells. Chemotherapy even more efficiently sensitized cells to the DR5-specific mutant variant of TRAIL DR5-B, which does not have an affinity for decoy receptors. Bortezomib and doxorubicin greatly enhanced the surface expression of the death receptors DR5 and DR4, while panobinostat increased expression of DR5 and suppressed expression of DR4 in both cell lines. All drugs increased surface expression of the decoy receptors DcR1 and DcR2. Unlike the combined treatment, if the cells were pretreated with chemotherapy for 24 h, the cytotoxic activity of TRAIL was less pronounced, while sequential treatment of cells enhanced the effectiveness of DR5-B. The same results were obtained with agonistic anti-DR5 antibodies. Thus, the effectiveness of TRAIL was rather limited due to changes in the ratio of death and decoy receptors and DR5-specific agonists may be preferred in combination antitumor therapy regimens.

A sample-type protein monolayer, that can be a stepping stone to practical devices, can behave as an electrically driven switch. This feat is achieved using a redox protein, cytochrome C (CytC), with its heme shielded from direct contact with the solid-state electrodes. Ab initio DFT calculations, carried out on the CytC-Au structure, show that the coupling of the heme, the origin of the protein frontier orbitals, to the electrodes is sufficiently weak to prevent Fermi level pinning. Thus, external bias can bring these orbitals in and out of resonance with the electrode. Using a cytochrome C mutant for direct S-Au bonding, approximately 80 % of the Au-CytC-Au junctions show at greater than 0.5 V bias a clear conductance peak, consistent with resonant tunneling. The on-off change persists up to room temperature, demonstrating reversible, bias-controlled switching of a protein ensemble, which, with its built-in redundancy, provides a realistic path to protein-based bioelectronics., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I-->N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I-->N transition., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.

Prefibrillar cytotoxicity was suggested as a common amyloid characteristic. We showed two types of albebetin prefibrillar oligomers are formed during incubation at pH 7.3. Initial round-shaped oligomers consist of 10-15 molecules determined by atomic force microscopy, do not bind thioflavin-T and do not affect viability of granular neurons and SH-SY5Y cells. They are converted into ca. 30-40-mers possessing cross-beta-sheet and reducing viability of neuronal cells. Neither monomers nor fibrils possess cytotoxicity. We suggest that oligomeric size is important for stabilising cross-beta-sheet core critical for cytotoxicity. As albebetin was used as a carrier-protein for drug delivery, examination of amyloidogenicity is required prior polypeptide biomedical applications.

Cytotoxicity of plasma free platelets or peripheral blood mononuclear cells (PBMCs) upon stimulation with Ca-ionophore A23187 at various (10(-9) - 10(-6) M) concentrations has been studied on human erythroid leukaemia cell line (K562). A 14 kDa protein, which did not display any cytotoxic activity to K562 cells, but stimulate PBMC proliferation has been isolated from the supernatant of plasma free platelets stimulated by 10(-7) M Ca-ionophore. The protein displayed a very slight mitogenic effect on PBMCs, but it enhanced proliferative response of PBMC stimulated by concanavalin A at suboptimal concentrations. The p14 N-terminal sequence ((1)VLERTXA(7)-) is identical to the region 99-103 residues of the human MHC class II antigen DQ-beta chain. Also, we identified this 14 kDa protein in the supernatant of PBMCs stimulated by 10(-7) M Ca-ionophore. Its N-terminal sequence (VLERTXA-) is identical to the one of p14 from the stimulated plasma-free platelet supernatant.

Human PBMCs were stimulated by ricin at very low concentrations (10(-14) M and lower) for 48 h at 37 degrees C. Cytolytic activity to K562 cell line and lymphokine secretion were been measured. PBMCs possessed the highest cytotoxicity (63 +/- 2.8% and 64 +/- 3.1%, respectively) to K562 cell line in the presence of 10(-15) M or 10(-16) M ricin. The high secretion of IL-1beta (2000 pg/ml), IL-2 (1300 pg/ml) and TNF-alpha (2200 pg/ml) was detected in the presence of 10(-15) M ricin. The mechanisms of the target cell death induced by PBMCs after co-incubation with various lectins: 10(-8) M phytohemagglutinin, 10(-8) M concanavalin A and ricin at 10(-15) M or 10(-16) M concentrations were compared. Internucleosomal DNA fragmentation characteristic of apoptotic mechanisms of the target cell death in all cases were observed, and the maximum of DNA fragmentation was registered in the presence of 10(-15) M ricin.

The cytolytic activity of human plasma free platelets treated with various stimulators (Ca-ionophore, PAF (platelet activating factor), PHA, and ricin) has been studied on human erythroid leukemia cell line (K562). The 14 kDa and 28 kDa platelet cytotoxic factors (PCF) have been purified from the supernatant obtained from platelets stimulated by 10(-7) M Ca-ionophore. The PCF-14 N-terminal sequence ((1)YAPQXQFGP(9)-) appeared to be homologous to the region 241-249 residues of the human C1s complement component. The PCF-28 N-terminal sequence (DVGLT-) did not display any homology to known human proteins. The PCF-14 and the PCF-28 have been detected both in the supernatants of platelets treated with various stimulators (10(-7) M Ca-ionophore A23187, or 10(-8) M PAF, or 10(-9) M PHA, or 10(-13) M ricin) and in the extracts of disrupted platelets treated by these stimulators. All of these stimulators enhanced the production of thromboxane A(2) by platelets, as evidenced from the accumulation of thromboxane B(2), a spontaneous hydrolysis product of thromboxane A(2). These results indicate that platelet cytotoxicity to the K562 cells and the release of PCF-14 and PCF-28 might be mediated by the stimulation of thromboxane A(2) synthesis, when platelets have been activated through the cycloxygenase pathway.