Seed storage protein variation in Indian barley using 2-D diagonal gel electrophoresis approach

Barley is the fourth most important cereal, which is used as feed and fodder. It is also used as raw material in the brewing industry and is rich in soluble dietary fiber. Thus, it has economic as well as health benefits. The present study studied seed storage protein characterization of malt, feed, two row, six row, salinity tolerant and susceptible Indian barley genotypes. The polymorphic bands in feed and malt barley were 59% and 54%; in two and six-row barley 35% and 59%; and in salt-tolerant and susceptible barley 38% and 59%. The salt-tolerant lines DL 88 and NB 1were exhibited five interlinked polypeptides, while NDB 1173, NB 3, and RD 2552 exhibited four, two, and one interlinked disulfide linkage, respectively. Whereas, in the case of salt susceptible lines Alfa 93, NB 2, K 508, BH 393 and K 551 a total of six each in Alfa 93 and K 551 barley lines, while in K 508 BH 393 barley lines, two disulfide-linked polypeptides were found. The current study portrayed a simple and economical procedure to characterize the seed protein by gel electrophoresis. Characterization of seed storage proteins by banding pattern analysis using one dimensional SDS-PAGE is the simple molecular technique to evaluate the barley lines with particular traits such as salinity tolerance ability, malt or feed quality, and others. The unique protein bands can also be used as a marker to identify the lines.

Green self-immolative polymer: molecular antenna to collect and propagate the signal for zymogen activation

Chemical zymogens of three different types were established herein around protein cysteinome, in each case converting the protein thiol into a disulfide linkage: zero length Z0, polyethylene glycol based ZPEG, and ZLA that features a fast-depolymerizing fuse polymer. The latter was a polydisulfide based on a naturally occurring water-soluble lipoic acid. Three zymogen designs were applied to cysteinyl proteases and a kinase and in each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only ZLA could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the enzyme active site. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. For ZLA, we also document a "chain transfer" bioconjugation mechanism and a unique zymogen exchange reaction between two proteins.

Albumin-Based Nanoparticles for the Delivery of Doxorubicin in Breast Cancer

Albumin-based nanoparticles are an emerging platform for the delivery of various chemotherapeutics because of their biocompatibility, safety, and ease of surface modification for specific targeting. The most widely used method for the preparation of albumin nanoparticles is by desolvation process using glutaraldehyde (GLU) as a cross-linker. However, limitations of GLU like toxicity and interaction with drugs force the need for alternative cross-linkers. In the present study, several cross-linking systems were evaluated for the preparation of Bovine Serum Albumin (BSA) nanoparticles (ABNs) encapsulating Doxorubicin (Dox). Based on the results obtained from morphological characterization, in vitro release, and therapeutic efficacy in cells, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)-modified ABNs (ABN-SPDP) was chosen. Since ABN-SPDP are formed with disulfide linkage, the drug release is facilitated under a highly reducing environment present in the tumor sites. The cytotoxicity studies of those ABN-SPDP were performed in three different breast cell lines, highlighting the mechanism of cell death. The Dox-encapsulated ABN-SPDP showed toxicity in both the breast cancer cells (MCF-7 and MDA-MB-231), but, remarkably, a negligible effect was observed in non-tumoral MCF-10A cells. In addition to the hydrophilic Dox, this system could be used as a carrier for hydrophobic drugs like SN38. The system could be employed for the preparation of nanoparticles based on human serum albumin (HSA), which further enhances the feasibility of this system for clinical use. Hence, the albumin nanoparticles developed herein present an excellent potential for delivering various drugs in cancer therapy.

Structure and antimicrobial activity of NCR169, a nodule-specific cysteine-rich peptide of Medicago truncatula

A model legume, Medicago truncatula, has over 600 nodule-specific cysteine-rich (NCR) peptides required for symbiosis with rhizobia. Among them, NCR169, an essential factor for establishing symbiosis, has four cysteine residues that are indispensable for its function. However, knowledge of NCR169 structure and mechanism of action is still lacking. In this study, we solved two NMR structures of NCR169 caused by different disulfide linkage patterns. We show that both structures have a consensus C-terminal β-sheet attached to an extended N-terminal region with dissimilar features; one moves widely, whereas the other is relatively stapled. We further revealed that the disulfide bonds of NCR169 contribute to its structural stability and solubility. Regarding the function, one of the NCR169 oxidized forms could bind to negatively charged bacterial phospholipids. Furthermore, the positively charged lysine-rich region of NCR169 may be responsible for its antimicrobial activity against Escherichia coli and Sinorhizobium meliloti. This active region was disordered even in the phospholipid bound state, suggesting that the disordered conformation of this region is key to its function. Morphological observations suggested the mechanism of action of NCR169 on bacteria. The present study on NCR169 provides new insights into the structure and function of NCR peptides.

A Crustin from Hydrothermal Vent Shrimp: Antimicrobial Activity and Mechanism

Crustin is a type of antimicrobial peptide and plays an important role in the innate immunity of arthropods. We report here the identification and characterization of a crustin (named Crus1) from the shrimp Rimicaris sp. inhabiting the deep-sea hydrothermal vent in Manus Basin (Papua New Guinea). Crus1 shares the highest identity (51.76%) with a Type I crustin of Penaeus vannamei and possesses a whey acidic protein (WAP) domain, which contains eight cysteine residues that form the conserved ‘four-disulfide core’ structure. Recombinant Crus1 (rCrus1) bound to peptidoglycan and lipoteichoic acid, and effectively killed Gram-positive bacteria in a manner that was dependent on pH, temperature, and disulfide linkage. rCrus1 induced membrane leakage and structure damage in the target bacteria, but had no effect on bacterial protoplasts. Serine substitution of each of the 8 Cys residues in the WAP domain did not affect the bacterial binding capacity but completely abolished the bactericidal activity of rCrus1. These results provide new insights into the characteristic and mechanism of the antimicrobial activity of deep sea crustins.

S-nitrosylated TDP-43 triggers aggregation, cell-to-cell spread, and neurotoxicity in hiPSCs and in vivo models of ALS/FTD

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of “sporadic” cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.

Biocompatible nucleus-targeted graphene quantum dots for selective killing of cancer cells via DNA damage

Graphene quantum dots (GQDs) are nano-sized graphene slices. With their small size, lamellar and aromatic-ring structure, GQDs tend to enter into the cell nucleus and interfere with DNA activity. Thus, GQD alone is expected to be an anticancer reagent. Herein, we developed GQDs that suppress the growth of tumor by selectively damaging the DNA of cancer cells. The amine-functionalized GQDs were modified with nucleus targeting TAT peptides (TAT-NGs) and further grafted with cancer-cell-targeting folic acid (FA) modified PEG via disulfide linkage (FAPEG-TNGs). The resulting FAPEG-TNGs exhibited good biocompatibility, nucleus uptake, and cancer cell targeting. They adsorb on DNA via the π–π and electrostatic interactions, which induce the DNA damage, the upregulation of the cell apoptosis related proteins, and the suppression of cancer cell growth, ultimately. This work presents a rational design of GQDs that induce the DNA damage to realize high therapeutic performance, leading to a distinct chemotherapy strategy for targeted tumor therapy.

Synthesis of Fucose Derivatives with Thiol Motifs towards Suicide Inhibition of Helicobacter pylori

The syntheses of six thiol-exhibiting monosaccharides towards suicide inhibition of Helicobacter pylori are reported. Blood group Antigen Binding Adhesin (BabA), a bacterial membrane-bound lectin, binds to human ABO and Lewis b blood group structures displayed on the surface of host epithelial cells. Crystal structures of the carbohydrate-recognition domain revealed a conserved disulfide bonded loop that anchors a critical fucose residue in these blood group structures. Disruption of this loop by N-acetylcysteine results in reduced BabA-mediated adherence to human gastric tissue sections and attenuated virulence in Lewis b-expressing transgenic mice. With a view of creating specific inhibitors of the lectin, we designed and successfully synthesised six fucose-derived compounds with thiol motifs to engage in a thiol-disulfide exchange with this disulfide bond of BabA and form a glycan-lectin disulfide linkage. Branching and extending the fucose backbone with 2- and 3-carbon thiol motifs delivered a range of candidates to be tested for biological activity against BabA.