Protein and Peptide Letters - Volume 22, Issue 2, 2015

Plant cystatins, also called phytocystatins, constitute a family of specific cysteine protease inhibitors found in several monocots and dicots, where they can be involved in the regulation of several endogenous processes and in defense against pests and pathogens, as well as in response to abiotic stress. In this mini-review we aimed to present isolated and characterized phytocystatins with potential use in control of plant disease caused by fungi.

During the past several years, studies on the protein aggregation process in the presence of cosolvents/ co-solutes have been looked into which provides significant insight in the stability of proteins in a crowded cellular milieu. Here, in the present report we have investigated the fibrillation of human serum albumin (HSA) under the mixed aqueous-ethanol solvent conditions at two different temperatures (37 °C and 65 °C). Self-association of protein was monitored using various spectroscopic and microscopic techniques. Results obtained from detailed investigation have shown that fibrillation of human serum albumin is favored at higher temperature (65 °C) at lower ethanol concentration. However, at 37 °C comparatively higher ethanol concentration is the prerequisite condition for fibrillation process to take place.

The N-terminus of the human dihydroorotate dehydrogenase (HsDHODH) has been described as important for the enzyme attachment in the inner mitochondrial membrane and possibly to regulate enzymatic activity. In this study, we synthesized the peptide acetyl-GDERFYAEHLMPTLQGLLDPESAHRL AVRFTSLGamide, comprising the residues 33-66 of HsDHODH N-terminal conserved microdomain. Langmuir monolayers and circular dichroism (CD) were employed to investigate the interactions between the peptide and membrane model, as micelles and monolayers of the lipids phosphatidylcholine (PC), 3-phosphatidylethanolamine (PE) and cardiolipin (CL). These lipids represent the major constituents of inner mitochondrial membranes. According to CD data, the peptide adopted a random structure in water, whereas it acquired α-helical structures in the presence of micelles. The π–A isotherms and polarization- modulated infrared reflection-absorption spectroscopy on monolayers showed that the peptide interacted with all lipids, but in different ways. In DPPC monolayers, the peptide penetrated into the hydrophobic region. The strongest initial interaction occurred with DPPE, but the peptide was expelled from this monolayer at high surface pressures. In CL, the peptide could induce a partial dissolution of the monolayer, leading to shorter areas at the monolayer collapse. These results corroborate the literature, where the HsDHODH microdomain is anchored into the inner mitochondrial membrane. Moreover, the existence of distinct conformations and interactions with the different membrane lipids indicates that the access to the enzyme active site may be controlled not only by conformational changes occurring at the microdomain of the protein, but also by some lipid-protein synergetic mechanism, where the HsDHODH peptide would be able to recognize lipid domains in the membrane.

Leishmaniasis is a wide spread tropical disease caused by protozoan parasite Leishmania which belongs to order kinetoplastida and family trypanosomatidae. Ornithine decarboxylase is key enzyme in polyamine biosynthesis in Leishmania donovani. Here, we report biochemical characterization of ornithine decarboxylase from L. donovani. Furthermore, we have also investigated the role of N-terminal extension (250 amino acids) found in ornithine decarboxylase of L. donovani (LdODC). The removal of N-terminal extended region of LdODC results in improved stability of the protein. However, the truncated LdODC does not show any activity. Apparently, while N-terminal extended region of LdODC helps in proper folding of the protein for catalytic activity, there is a stability trade-off. The native full length LdODC with N-terminal extension has activity but lower stability. Thus, there is trade off of conformational stability for enzyme activity. Comparison of biochemical properties of both, fulllength and truncated enzymes, have provided interesting insights about the role of N-terminal extension in the protein.

Synthesis of α2,8-polysialic acid (polySia) glycans are catalyzed by two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), which are two members of the ST8Sia gene family of sialytransferases. During polysialylation, both STX and PST catalyze the transfer of multiple Sia residues from the activated sugar nucleotide precursor, CMP-Neu5Ac (Sia), to terminal Sia residues on N- and Olinked oligosaccharide chains on acceptor glycoproteins, including the neural cell adhesion molecule (NCAM), which is the major carrier protein of polySia. Based on our new findings and previously published studies, this review summarizes the present concepts regarding the molecular mechanism underlying regulation of protein-specific polysialylation of NCAM that includes the following: (1) Determination of the catalytic domains and specific regions within ST8Sia IV for recognizing and catalyzing the efficient polysialylation of NCAM; (2) Identification of key amino acid residues within the PSTD motif of ST8Sia IV that are essential for polysialylation; (3) Verification of key amino acids in the PBR domain of ST8Sia IV required for NCAM-specific polysialylation; and (4) a 3D conformational study of ST8Sia IV based on the Phyre2 server to discover the relationship between the structure and its functional domains of the polyST. Based on these results, our 3D model of ST8Sia IV was used to identify and characterize the catalytic domains and amino acid residues critical for catalyzing polysialylation, and have provided new structural information for supporting a detailed mechanism of polyST-NCAM interaction required for polysialylation of NCAM, findings that have not been previously reported.

The overwhelming demand for food requires the application of technology on field. An important issue that limits the productivity of crops is related to insect attacks. Hence, several studies have evaluated the application of different compounds to reduce the field losses, especially insecticide compounds from plant sources. Among them, plant protease inhibitors (PIs) have been studied in both basic and applied researches, displaying positive results in control of some insects. However, certain species are able to bypass the insecticide effects exerted by PIs. In this review, we disclosed the adaptive mechanisms showed by lepidopteran and coleopteran insects, the most expressive insect orders related to crop predation. The structural aspects involved in adaptation mechanisms are presented as well as the newest alternatives for pest control. The application of biotechnological tools in crop protection will be mandatory in agriculture, and it will be up to researchers to find the best candidates for effective control in long-term.

Antimicrobial peptides (AMPs) represent a large and ubiquitous group of peptides. The current crisis in antibiotic therapy has led to an intensified search for new antimicrobial agents. In this regard, scorpion venom constitutes a rich source of biologically active peptides including AMPs. In the present study, the purification of a novel peptide with antimicrobial activity against the Gram-negative bacteria Klebsiella pneumoniae is described. This antimicrobial peptide, named Cm38, was purified from Centruroides margaritatus scorpion venom using a two-step chromatographic strategy using C8 and C18 columns. This toxin inhibits the proliferation of the Gram-negative bacteria Klebsiella pneumoniae with a Minimal Inhibitory Concentration (MIC) of 64 μM. An analysis of the N-terminal sequence of Cm38 revealed a close structural relationship to Cn11, a Na+-channel modulator toxin previously isolated from Centruroides noxius scorpion venom. Therefore, to test Cm38 for effects on ion channels, we measured its effects on action potential firing in cultured dorsal root ganglion neurons. Cm38 depolarized and increased action potential firing in a subset of neurons tested. The present work reports a new peptide related to Cn11 with antimicrobial properties that is also active in neurons.

Geobacillus zalihae sp. nov., which produces a putative thermostable lipase, represents a novel species, with type strain T1. The characterisation of this intrinsically thermostable T1 lipase either physicochemically or structurally is an important task. The crystallisation of T1lipase in space was carried out using a High-Density Protein Crystal Growth (HDPCG) apparatus with the vapour diffusion method, and X-ray diffraction data were collected. The microgravity environment has improved the size and quality of the crystals as compared to earth grown crystal. The effect of microgravity on the crystallisation of T1 lipase was clearly evidenced by the finer atomic details at 1.35 A resolution. Better electron densities were observed overall compared with the Earth-grown crystals, and comparison shows the subtle but distinct conformations around Na+ ion binding site stabilized via cation-π interactions. This approach could be useful for solving structure and function of lipases towards exploiting its potentials to various industrial applications.

Mesial temporal lobe epilepsy (MTLE) is the most prevalent form of refractory symptomatic epilepsy, most patients with MTLE respond poorly to anti-epileptic drugs (AEDs). The pathophysiological mechanism of MTLE remains unclear, blocking the path to successful prevention and treatment. We know that change in brain plasticity after long epileptic seizures in early childhood can produce long-term effects in the individual. It is a dynamic process, developing from early injuries in the immature brain to chronic epilepsy in adults. However, most MTLE studies are limited to a certain aspect of a certain time, rather than taking the long view of epilepsy as a systematic and chronological disease. In this report, we screened and identified proteins that are differentially expressed in the acute, latent and chronic stages of MTLE in an immature rat model. Proteins were screened by proteomics, and western blot was used to confirm differentially expressed proteins, such as synapsin-1, dynamin-1 and neurogranin, in hippocampi from both rats and humans with MTLE. Our results show that the synaptic dysfunction caused by abnormal expression of synaptic proteins play a critical role in the pathogenesis of chronic MTLE.

Gastric cancer is a major cause of mortality and morbidity around world. However the effectiveness of the current approaches to the diagnosis and treatment of gastric cancer is limited. Recombinant targeted toxins may represent a novel direction of cancer therapy. In this study, we aimed to explore whether recombinant toxins fused with the truncated forms of G17 could target to kill cancer cells by recognizing CCK2R. Four recombinant Pseudomonas toxins PE38 fused with the forward or reverse truncated forms of G17 (G14 and G13) were successfully constructed, expressed, and purified. Their characteristics were further analyzed by SDS-PAGE, western blot and indirect immunofluorescence assay. The cytotoxicity assay demonstrated that only reversely fused recombinant toxins rG14PE38 and rG13PE38 exhibited certain toxicity on several cancer cell lines, and a competition assay indicated that the binding of the reverse gastrin-endotoxin to CCK2R (+) cells may be mediated by interaction between gastrin/gastrin-like and CCK2R.