Robert Nawrot

Plant antimicrobial peptides

Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.

Nucleases isolated from Chelidonium majus L. milky sap can induce apoptosis in human cervical carcinoma HeLa cells but not in Chinese Hamster Ovary CHO cells.

Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milk sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the dependence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 +/- 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 +/- 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflammatory reaction.

Viral and Other Cell-Penetrating Peptides as Vectors of Therapeutic Agents in Medicine

Efficient delivery of heterologous molecules for treatment of cells is a great challenge in modern medicine and pharmacology. Cell-penetrating peptides (CPPs) may improve efficient delivery of a wide range of macromolecular cargos, including plasmid DNA, small interfering RNA, drugs, nanoparticulate pharmaceutical carriers, and anticancer drugs. In this paper, we present the history of CPPs’ discovery with special attention drawn to sequences of viral origin. We also describe different CPP families with regard to their physicochemical properties and numerous mechanisms of CPP cell uptake by direct penetration and endocytotic pathways. A detailed description is focused on formation of carrier-cargo complexes, which are needed for practical use of CPPs in medicine and biotechnology. Examples of successful application of CPPs in treatment of human diseases are also presented, including decreased tumor growth and induction of cancer cell death. Finally, we review modern design approaches to novel CPPs and prediction of their activity. To sum up, the current review presents a thorough and up-to-date knowledge of CPPs and may be a valuable source of information for researchers in pharmacology designing new therapeutic agents.

Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

BackgroundCorydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine), belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers.MethodsNucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS).ResultsThe studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml) - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR) proteins.ConclusionsThe cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.

Global proteomic analysis of Chelidonium majus and Corydalis cava (Papaveraceae) extracts revealed similar defense-related protein compositions

Chelidonium majus and Corydalis cava are phylogenetically closely related (Papaveraceae family). The medicinal and pharmaceutical interest in these plants is based on their synthesis of pharmaceutically important compounds, such as alkaloids, flavonoids, phenolic acids and proteins. C. majus shoot and C. cava tuber extracts have been used in traditional folk medicine to treat many diseases, such as fungal, bacterial and viral infections, liver disorders, fever, post-traumatic, colic, abdominal and menstrual pains and even cancer. This study attempts to perform a global comparative proteomic analysis of pharmacologically important extracts from these two closely related unsequenced plant species to gain insights into the protein basis of these plant organs and to compare their common and specific proteomic compositions. We used a shotgun proteomic approach combined with label-free protein quantitation according to the exponentially modified protein abundance index (emPAI). In total, a mean number of 228 protein identification results were recorded in C. cava tuber extracts and about 1240 in C. majus shoot extracts. Comparative analysis revealed a similar stress and defense-related protein composition of pharmacologically active plant species and showed the presence of different pathogenesis-related and low molecular inducible antimicrobial peptides. These findings could form the basis for further elucidation of the mechanism of the strong pharmacological activities of these medicinal plant extracts.

Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown pathology, involving reproductive and metabolic abnormalities. Oocyte-specific genes are a group of genes expressed exclusively in ovarian tissue; therefore, they can play an important role in ovarian pathologies such as PCOS. The zona pellucida 4 (ZP4) gene encodes glycoprotein which is a part of the extracellular matrix of oocyte.Materials and methodsWe analyzed 87 patients with PCOS, which were divided into four groups depending on their phenotype. In each patient, we performed profound clinical and biochemical analysis, including the measurement of serum androgens. The ovarian tissue samples were used to perform a real-time polymerase chain reaction and immunohistochemical staining using anti-ZP4 monoclonal antibodies. The ZP4 gene was sequenced from peripheral lymphocytes.ResultsThe expression of ZP4 was present in early antral follicles and was stronger in mature follicles. The subgroup of patients with eumenorrhea and without hyperandrogenism presented the highest expression of ZP4 in ovarian tissue. In one case, we found a mutation of the ZP4 gene. No correlations were found between the ZP4 expression level and biochemical or clinical indices.ConclusionsData from this and animal studies suggest a possible relationship between androgens and ZP4 expression. ZP4 expression is highest among patients with PCOS and a regular cycle, and this is a consequence of the presence of mature follicles in this group. In some patients with PCOS and infertility, ZP4 mutation can be found.

Incorrectly annotated keratin derived peptide sequences lead to misleading MS/MS data interpretation

In the course of the nanoLC-nanoESI-MS/MS analysis of Chelidonium majus proteins we detected an extremely abundant 12 aa peptide (sequence: TNAENEFVTIKK). The Mascot search against NCBInr database with Viridiplantae taxonomic restriction revealed its complete identity to Unknown protein 18 from Pseudotsuga menziesii (P85925). The same sequence has also been submitted as Unknown protein 1 (P86104) from Vitis rotundifolia and as a part of the oxygen-evolving enhancer protein 1 (OEE1) from Pinus strobus (P84718). After careful inspection the record P84718 turned out to comprise a set of peptides of unconfirmed origin rather than complete protein sequence. In this paper we present extensive data indicating that the peptide in question may originate from type II cytoskeletal keratin - a common contaminant in protein samples. We found empirical evidence that it can be detected in several types of keratin-contaminated samples and its sequence is identical to one of the proteotypic peptides commonly observed for keratins. Nevertheless, the peptide has been annotated as plant protein and thus leads to data misinterpretation. We advise extreme caution when dealing with sequences of Unknown protein 18 (P. menziesii), Unknown protein 1 (V. rotundifolia) and OEE1 (P. strobus) since they are at best poorly annotated, if not artifactual. Biological significance To our knowledge, this is the first report indicating that the peptide TNAENEFVTIKK, which is identical to Unknown protein 18 from P. menziesii (P85925), Unknown protein 1 (P86104) from V. rotundifolia and a part of the oxygen-evolving enhancer protein 1 (OEE1) from P. strobus (P84718), may originate from type II cytoskeletal keratin - a common contaminant in protein samples. We found empirical evidence that it can be detected in several types of keratin-contaminated samples and its sequence is identical to one of the proteotypic peptides commonly observed for keratins. Nevertheless, the peptide has been annotated as plant protein and thus, without proper quality assessment, leads to biological misinterpretation of proteomic data.

Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera).

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole-larval preparations, permitting studies of the very small whole-larval preparations. Protein preparations were analyzed using FITC-casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0-4.0). Cysteine protease activity increased at acidic pH and in the presence of β-mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe(2+) , Ca(2+) , Mg(2+) , Zn(2+) , and K(+) (10 mM). Fe(2+) and Zn(2+) significantly decreased enzyme activity at all pH values, Ca(2+) and Mg(2+) at pH 6.2 and Mg(2+) at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N-p-tosyl-l-phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, Nα -tosyl-l-lysine chloromethyl ketone hydrochloride, N-p-tosyl-l-phenylalanine chloromethyl ketone, trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin-like) proteases and metalloproteases act in cereal leaf beetle digestion.

Isolation and characterization of a non-specific lipid transfer protein from Chelidonium majus L. latex

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins, which mostly play a role in intracellular lipid transport and antimicrobial defense. Recently it was shown using shotgun proteomic approach that the whole plant extract of Chelidonium majus L. (Papaveraceae) contains relatively abundant nsLTPs. Therefore the aim of the work was to isolate and characterize nsLTP from C. majus latex. Results obtained using PCR approach with degenerate primers showed the presence of nsLTP protein in C. majus root latex, named CmLTP 9.5. The protein consists of 93 aa with a molecular weight of 9.5kDa (NCBI GenBank accession no. ALT21495, coded by KP733898). The mature form of CmLTP 9.5 has a molecular weight of 7.147kDa and contains typical eight strictly conserved cysteine residues. A 3D model of CmLTP 9.5 displays a hydrophobic cavity. The isolated protein fraction tested using diffusion method and critical dilution assay showed strong antibacterial activity towards Gram-negative Campylobacter jejuni as well as Gram-positive Listeria greyi and Clostridium perfringens. Further studies using protein expression system are required to fully understand CmLTP 9.5 mode of action.