Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry.

OBJECTIVES: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5. RESULTS: We detected more than 100 monoisotopic masses corresponding to thymosin ß4 and truncated forms of ubiquitin, prothymosin α, thymosin ß4, and thymosin ß9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3. CONCLUSION: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.

Analysis of hepatitis B virus preS1 variability and prevalence of the rs2296651 polymorphism in a Spanish population.

AIM: To determine the variability/conservation of the domain of hepatitis B virus (HBV) preS1 region that interacts with sodium-taurocholate cotransporting polypeptide (hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism (S267F, NTCP variant) in a Spanish population. METHODS: Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection (CHB) (n = 41, 73% Caucasians), patients with resolved HBV infection (n = 100, 100% Caucasians) and an HBV-uninfected control group (n = 105, 100% Caucasians). Variability/conservation of the amino acid (aa) sequences of the NTCP-interacting domain, (aa 2-48 in viral genotype D) and a highly conserved preS1 domain associated with virion morphogenesis (aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis. RESULTS: The HBV preS1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21 (in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCP-interacting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies (25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms (34% vs 27.3%), according to consensus sequences from GenBank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable (limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant. CONCLUSION: In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null.

To elute or not to elute in immunocapture bottom-up LC-MS.

Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested.

Peptidomics of the zebrafish Danio rerio: In search for neuropeptides.

(Neuro)peptides are small messenger molecules that are derived from larger, inactive precursor proteins by the highly controlled action of processing enzymes. These biologically active peptides can be found in all metazoan species where they orchestrate a wide variety of physiological processes. Obviously, detailed knowledge on the actual peptide sequences, including the potential existence of truncated versions or presence of post-translation modifications, is of high importance when studying their function. A peptidomics approach therefore aims to identify and characterize the endogenously present peptide complement of a defined tissue or organism using liquid chromatography and mass spectrometry. While the zebrafish Danio rerio is considered as an important aquatic model for medical research, neuroscience, development and ecotoxicology, very little is known about their peptidergic signaling cascades. We therefore set out to biochemically characterize endogenously present (neuro)peptides from the zebrafish brain. This peptidomics setup yielded >60 different peptides in addition to various truncated versions. SIGNIFICANCE: Though the zebrafish is a well-established model organism to study vertebrate biology and gene functions in either a medical or (eco)toxicological context, very little knowledge about neuropeptidergic signaling cascades is available. We therefore set out to characterize endogenously present peptides from the zebrafish brain using a peptidomics setup yielding a total number of 105 peptide identifications. To our knowledge, it is the first attempt to biochemically isolate and characterize neuropeptides from a fish species in a high-throughput manner. This archive of identified endogenous peptides is likely to aid further functional elucidation of defined neuropeptidergic signaling systems (e.g. characterization of cognate G-protein coupled receptors). Furthermore, our methodology allows studying the changes in peptide expression in response to changes in the organism or the environment using differential peptidomics.

A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55Gag polyprotein alters nucleic acid binding properties.

HIV Gag (Pr55Gag), a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Pr55Gag has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (Δp6). In vivo, p6 is crucial to the budding process and interacts with protein complexes in the ESCRT (Endosomal Sorting Complexes Required for Transport) pathway, it has been difficult to study its role in the context of Gag using in vitro approaches. Here we report the generation of a full length Gag construct containing a tobacco etch virus (TEV)-cleavable C-terminal hexahistidine tag, allowing a detailed comparison of its nucleic acid binding properties with other constructs, including untagged, Δp6, and C-terminally tagged (TEV-cleavable and non-cleavable) Gags, respectively. We have developed a standard expression and purification protocol that minimizes nucleic acid contamination and produces milligram quantities of full length Gag for in vitro studies and compound screening purposes. We found that the presence of a carboxyl-terminal hexahistidine tag changes the nucleic binding properties compared to the proteins that did not contain the tag (full length protein that was either untagged or reulted from removal of the tag during purification). The HIV Gag expression and purification protocol described herein provides a facile method of obtaining large quantities of high quality protein for investigators who wish to study the full length protein or the effect of the p6 domain on the biophysical properties of Gag.

HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology.

Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease.

Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide.

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth, and its activity is suppressed by MSTN propeptide (MSTNpro), the N-terminal part of MSTN precursor cleaved during post-translational MSTN processing. The current study examined which region of flatfish (Paralichthys olivaceus) MSTN-1 propeptide (MSTN1pro) is critical for MSTN inhibition. Six different truncated forms of MSTN1pro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in Escherichia coli, and partially purified by an affinity chromatography for MSTN-inhibitory activity examination. Peptides covering different regions of flatfish MSTN1pro were also synthesized for MSTN-inhibitory activity examination. A MBP-fused MSTN1pro region consisting of residues 45-100 had the same MSTN-inhibitory potency as the full sequence flatfish MSTN1pro (residues 23-265), indicating that the region of flatfish MSTN1pro consisting of residues 45-100 is sufficient to maintain the full MSTN-inhibitory capacity. A MBP-fused MSTN1pro region consisting of residues 45-80 (Pro45-80) also showed MSTN-inhibitory activity with a lower potency, and the Pro45-80 demonstrated its MSTN binding capacity in a pull-down assay, indicating that the MSTN-inhibitory capacity of Pro45-80 is due to its binding to MSTN. Flatfish MSTN1pro synthetic peptides covering residues 45-65, 45-70, and 45-80 demonstrated MSTN-inhibitory activities, but not the synthetic peptide covering residues 45-54, indicating that residues 45-65 of flatfish MSTN1pro are essential for MSTN inhibition. In conclusion, current study show that like the mammalian MSTNpro, the MSTN-inhibitory region of flatfish MSTN1pro resides near its N-terminus, and imply that smaller sizes of MSTNpro can be effectively used in various applications designed for MSTN inhibition.

Functional Analysis of Periplakin and Envoplakin, Cytoskeletal Linkers, and Cornified Envelope Precursor Proteins.

Envoplakin and periplakin are the two smallest plakin family cytoskeletal linker proteins that connect intermediate filaments to cellular junctions and other membrane locations. These two plakins have a structural role in the assembly of the cornified envelope (CE), the terminal stage of epidermal differentiation. Analysis of gene-targeted mice lacking both these plakins and the third initial CE scaffold protein, involucrin, demonstrate the importance of the structural integrity of CE for a proper epidermal barrier function. It has emerged that periplakin, which also has a wider tissue distribution than envoplakin, has additional, independent roles. Periplakin participates in the cytoskeletal organization also in other tissues and interacts with a wide range of membrane-associated proteins such as kazrin and butyrophilin BTN3A1. This review covers methods used to understand periplakin and envoplakin functions in cell culture models, including siRNA ablation of periplakin expression and the use of tagged protein domain constructs to study localization and interactions. In addition, assays that can be used to analyze CEs and epidermal barrier function in gene-targeted mice are described and discussed.

Effective Strategies for Diagnosis of Systemic Inflammatory Response Syndrome (SIRS) due to Bacterial Infection in Surgical Patients.

Surgery associated with trauma and soft tissue injuries after surgery significantly activates the systemic immune response. If an infection after surgery occurs, the response is even stronger. Due to spontaneous activation of immune response and elevated biomarkers for sepsis and cytokines, posttraumatic complications such as new-coming postoperative infections are difficult to diagnose. Sepsis as systemic inflammatory response syndrome (SIRS) rapidly progresses through severe sepsis to septic shock and organ failure, and with no applied antibiotic treatment, the disease often ends at death of the patients. In the treatment of non-surgery patients, the biomarkers like white cell blood count, C-reactive protein (CRP) or procalcitonin (PCT) proved to be useful in sepsis recognition. However, diagnostics after surgeries are more complicated and these biomarkers are not ideal. The solution is a sepsis biomarker, which would have high sensitivity and specificity, that can improve diagnostic accuracy of sepsis, should also be measured easily by the patients, and should not be too expensive. We think more sensitive and specific biomarkers such as presepsin (sCD14-ST) or CD64 index on neutrophils could be useful. A diagnosis of sepsis should be based on clinical signs, and clinicians should use biomarker that is not only most sensitive and specific but also is cost effective. Furthermore, confirmation of the bacterial or fungal infection with blood cultures or with the use of broad range polymerase chain reaction (PCR), when culturing is impossible, should be performed.

Lung cancer may increase serum procalcitonin level.

INTRODUCTION: Serum procalcitonin (PCT) is a biomarker used routinely to diagnose infections. Some malignancies are usual false positives for PCT. However, its value and behavior in the setting of lung cancers are poorly known. The objective of this study was to assess PCT positivity in a lung cancer cases series. METHOD: Between November 2011 and September 2012, all cases of newly diagnosed lung cancer with a pre-antineoplastic PCT assay and no patent signs of infection were included in the study. All PCT levels were assessed by immunofluorescent assay in a single laboratory. RESULTS: Eighty-nine patients were included (70.8% male; mean age 62; small-cell cancer 20.2%; stage IV cancer 60.7%). Overall, PCT was positive in 42%. A neuroendocrine component, having 2 or more metastatic sites, having a pleura or a liver metastasis, and being positive for CRP were all significantly associated with positive PCT in univariate analysis. In multivariate analysis, only the presence of a neuroendocrine component remained strongly associated with a positive PCT (AOR=7.24 [CI=95% 1.91-27.51]; P=0.004). Finally, baseline PCT levels <0.5 µg/l were found in 43% of NSCLC with a neuroendocrine component, vs. 9% of cancers with other histology (P=0.0001). CONCLUSION: Lung cancer may cause false positives for procalcitonin, particularly in cases of neuroendocrine cancers or in the presence of multiple metastases. These results should be taken into account for PCT-based decisional algorithms.

Expression and characterization of myristoylated preS1-conjugated nanocages for targeted cell delivery.

Lipid modification of proteins plays key roles in cellular signaling pathways. We describe the development of myristoylated preS1-nanocages (myr-preS1-nanocages) that specifically target human hepatocyte-like HepaRG cells in which a specific receptor-binding peptide (preS1) is joined to the surface of naturally occurring ferritin cages. Using a genetic engineering approach, the preS1 peptide was joined to the N-terminal regions of the ferritin cage via flexible linker moieties. Myristoylation of the preS1 peptide was achieved by co-expression with yeast N-myristoyltransferase-1 in the presence of myristic acid in Escherichia coli cells. The myristoylated preS1-nanocages exhibited significantly greater specificity for human hepatocyte-like HepaRG cells than the unmyristoylated preS1-nanocages. These results suggest that the lipid-modified nanocages have great potential for effective targeted delivery to specific cells.

MixGF: spectral probabilities for mixture spectra from more than one peptide.

In large-scale proteomic experiments, multiple peptide precursors are often cofragmented simultaneously in the same mixture tandem mass (MS/MS) spectrum. These spectra tend to elude current computational tools because of the ubiquitous assumption that each spectrum is generated from only one peptide. Therefore, tools that consider multiple peptide matches to each MS/MS spectrum can potentially improve the relatively low spectrum identification rate often observed in proteomics experiments. More importantly, data independent acquisition protocols promoting the cofragmentation of multiple precursors are emerging as alternative methods that can greatly improve the throughput of peptide identifications but their success also depends on the availability of algorithms to identify multiple peptides from each MS/MS spectrum. Here we address a fundamental question in the identification of mixture MS/MS spectra: determining the statistical significance of multiple peptides matched to a given MS/MS spectrum. We propose the MixGF generating function model to rigorously compute the statistical significance of peptide identifications for mixture spectra and show that this approach improves the sensitivity of current mixture spectra database search tools by a ≈30-390%. Analysis of multiple data sets with MixGF reveals that in complex biological samples the number of identified mixture spectra can be as high as 20% of all the identified spectra and the number of unique peptides identified only in mixture spectra can be up to 35.4% of those identified in single-peptide spectra.

Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli.

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

Measurement of nonsulfated cholecystokinins.

Most proteins undergo posttranslational modifications that govern the function of the protein. In synchrony, correspondingly unmodified proteins that are functionally silent or act differently may also be synthesized. The gut hormone precursor, procholecystokinin (proCCK) is an example of a protein that is heavily modified. An essential modification is O-sulfation of Y77, which is necessary for the gallbladder emptying effect of CCK peptides via the CCKA-receptor. In order to examine possible in vivo synthesis also of nonsulfated CCK, we have established a two-step analysis that requires tryptic cleavage at a defined processing site in proCCK (R75-D76) followed by monospecific RIA-measurement of the then exposed nonsulfated N-terminal sequence of CCK-8 (DYMGW…). The analysis shows that endocrine cells in the gut synthesize nonsulfated CCK peptides (-58, -33, -22, and -8) in the order of 20-35% of the corresponding sulfated CCKs. Since nonsulfated CCK peptides are full agonists of the CCKB-receptor, the assay has revealed a hitherto unrecognized gut hormonal peptide system. The assay may prove useful in the diagnosis and control of diseases with hyperCCKemia. This includes CCK-producing neuroendocrine tumors such as the recently described CCKomas and medullary thyroid C-cell carcinomas.

Chronic nicotine treatment impacts the regulation of opioid and non-opioid peptides in the rat dorsal striatum.

The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.

Caerulein precursor fragment (CPF) peptides from the skin secretions of Xenopus laevis and Silurana epitropicalis are potent insulin-releasing agents.

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid clawed frog Xenopus laevis (Pipidae) led to the identification of 10 peptides with the ability to stimulate the release of insulin from the rat BRIN-BD11 clonal ß cell line. These peptides were purified to near homogeneity and structural characterization showed that they belong to the magainin (2 peptides), peptide glycine-leucine-amide (PGLa) (1 peptide), xenopsin precursor fragment (1 peptide), and caerulein precursor fragment (CPF) (6 peptides) families. CPF-1, CPF-3, CPF-5 and CPF-6 were the most potent producing a significant (P < 0.05) increase in the rate of insulin release at concentration of 0.03 nM. CPF-7 (GFGSFLGKALKAALKIGANALGGAPQQ) produced the maximum stimulation of insulin release (571 ± 30% of basal rate at 3 µM). In addition, CPF-SE1 (GFLGPLLKLGLKGVAKVIPHLIPSRQQ), previously isolated from skin secretions of the tetraploid frog Silurana epitropicalis, produced a significant (P < 0.05) increase in the rate of insulin release at 0.03 nM with a 514 ± 13% increase over basal rate at 3 µM. No CPF peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from BRIN-BD11 cells at concentrations up to 3 µM indicating that the integrity of the plasma membrane had been preserved. The mechanism of action of the CPF peptides involves, at least in part, membrane depolarization and an increase in intracellular Ca(2+) concentration. The CPF peptides show potential for development into agents for the treatment of Type 2 diabetes.

Cyclotide isolation and characterization.

Cyclotides are disulfide-rich cyclic peptides produced by plants with the presumed natural function of defense agents against insect pests. They are present in a wide range of plant tissues, being ribosomally synthesized via precursor proteins that are posttranslationally processed to produce mature peptides with a characteristic cyclic backbone and cystine knot motif associated with their six conserved cysteine residues. Their processing is not fully understood but involves asparaginyl endoproteinase activity. In addition to interest in their defense roles and their unique topologies, cyclotides have attracted attention as potential templates in peptide-based drug design applications. This chapter provides protocols for the isolation of cyclotides from plants, their detection and sequencing by mass spectrometry, and their structural analysis by NMR, as well as describing methods for the isolation of nucleic acid sequences that encode their precursor proteins. Assays to assess their membrane-binding interactions are also described. These protocols provide a "starter kit" for researchers entering the cyclotide field.

Molecular cloning of skin peptide precursor-encoding cDNAs from tibial gland secretion of the Giant Monkey Frog, Phyllomedusa bicolor (Hylidae, Anura).

The skins of phyllomedusine frogs have long been considered as being tremendously rich sources of bioactive peptides. Previous studies of both peptides and cloning of their precursor encoding cDNAs have relied upon methanolic skin extracts or the dissected skins of recently deceased specimens and have not considered the different glands in isolation. We therefore focused our attention on the tibial gland of the Giant Monkey Frog, Phyllomedusa bicolor and constructed a cDNA library from the skin secretion that was obtained via mechanical stimulation of this macrogland. Using shotgun cloning, four precursors encoding host-defense peptides were identified: two archetypal dermaseptins, a phyllokinin and a phylloseptin that is new for this species but has been recently described from the Waxy Monkey Leaf Frog, Phyllomedusa sauvagii. Our study is the first to report defensive peptides specifically isolated from anuran tibial glands, confirming the hypothesis that these glands also contribute to chemical defense. Moreover, the discovery of novel compounds for this otherwise very well characterized species suggests that this largely neglected gland might possess a different cocktail of secretions from glands elsewhere in the same animal. We will also discuss some evolutionary implications of our findings with respect to the adaptive plasticity of secretory glands.

Recombinant medaka (Oryzias melastigmus) pro-hepcidin: Multifunctional characterization.

Recently, two hepcidin variant genes (Om-hep1 and Om-hep2) were identified in a model fish marine medaka and both were highly induced in vivo with bacterial challenge, suggesting that the medaka hepcidin may have a similar function to other reported teleostean hepcidins. In the present study, the antibacterial, antiviral and antitumor activities of Om-hep1 were determined using its synthetic and recombinant pro-peptides. The recombinant pro-hepcidin1 was expressed in Escherichia coli and an effective method to produce recombinant Pro-Omhep1 was developed in order to obtain a right folded structure. The results showed that both the synthetic mature peptide and recombinant pro-peptide had similar antibacterial activity against Gram-positive and negative bacteria. In particular, both the synthetic mature Om-hep1 and recombinant Pro-Omhep1 inhibited the viral replication of white spot syndrome virus in the hematopoietic tissue cells of the crayfish Cherax quadricarinatus. Om-hep1 also presented antitumor activity on the cultured human hepatocellular carcinoma cells. In addition, the antimicrobial mechanism of Om-hep1 was measured and it was found that Om-hep1 was likely to be non-membranolytic. The recombinant Pro-Omhep1 performed better biological activity compared to the synthetic mature Om-hep1. This study suggested that Om-hep1 was likely to be an important multifunction protein involved in various resistance actions in the marine medaka immune system.

Conformational plasticity of proNGF.

Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor.