Protein and Peptide Letters - Volume 17, Issue 7, 2010

The cyclic peptide EGLNcΨ[CON((CH2)3NH)pYNleE(NHCH2CO)]L-NH2 (1) was designed and synthesized according to a native interaction partner of tyrosine phosphatase SHP-1. We introduced N-aminopropyl-phosphotyrosine to enable backbone-side chain cyclization with a glutamic acid derivative as counterpart for cyclization. Different approaches have been compared to find a strategy for the generation of backbone and backbone-side chain cyclic phosphopeptides.

A dimeric 34-kDa antifungal protein, designated as acafusin, was purified from Acacia confusa seeds using Q-Sepharose, DEAE-cellulose, Mono S and Superdex S75. It demonstrated antifungal activity toward Rhizoctonia solani with an IC50 of 28 μM. Acafusin inhibited the activity of HIV-1 reverse transcriptase mildly with an IC50 of 80 μM.

A 22.4-kDa dimeric hemagglutinin was isolated from tubers of Colocasia esculenta cv. ‘Small Taro’ by employing a purification protocol that involved ion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono Q, and FPLC-gel filtration on Superdex 75. The hemagglutinin was isolated from the fraction of the taro extract adsorbed on Q-Sepharose and subsequently adsorbed on Mono Q. The major absorbance peak from the Superdex 75 column constituted purified hemagglutinin. Its hemagglutinating activity could not be inhibited by simple sugars, and was stable after exposure for 30 minutes to temperatures up to 40°C and to ambient pH in the range of pH 2 to pH 13. The activity decreased progressively when the ambient temperature was raised from 40°C to 100°C. Negligible activity was detected at 100°C. The activity plummeted, with about 40% and 10% remaining, 4 minutes and 20 minutes after exposure to 100°C, respectively. About half of the activity remained at pH 0 and pH 1 whereas the activity was completely abolished at pH 14. The hemagglutinin exhibited slight anti-tumor activity toward hepatoma HepG2 cells, and weak mitogenic activity toward murine splenocytes. It induced expression of the cytokines interleukin- 1β, inteleukin-2, interferon-γ and tumor necrosis factor-α. However, it was devoid of anti-fungal activity toward a number of fungal species.

The crystal structure for the Deinococcus radiodurans Nudix protein DR_0079 was recently determined in the metal-free form at 1.9 Å resolution (2O5F). The protein adopts the fundamental fold common to the Nudix family of proteins, a large mixed β-sheet sandwiched between the α-helix of the “Nudix box” and a second α-helix. The protein's physical properties were further characterized by circular dichroism (CD) spectroscopy. A CD thermal melt at 220 nm indentifies an inflection point at ∼52°C. However, unlike typical CD thermal melts, the negative ellipticity at 220 nm becomes more negative upon passing through the inflection point. Both NMR spectroscopy and size exclusion chromatography indicate that heating effects the irreversible formation of a large molecular weight complex. After cooling, the negative ellipiticity at 220 nm increases further, and overall, the CD spectrum at 25°C suggests that heat-treated DR_0079 has more α-helical and β-sheet structure than non-heat treated DR_0079.

Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play key roles in the regulation of metabolic processes, reproduction, and defense against pathogens. The proteomics approach was used to identify proteins that are induced by JA and SA in the tomato cultivars Roma and Pant Bahr, which are susceptible and resistant to bacterial wilt, respectively. Threonine deaminase and leucine amino peptidase were upregulated, and ribulose-1,5-bisphosphate carboxylase/oxygenase small chain was downregulated by time-course application of JA. Translationally controlled tumor protein was upregulated by time-course application of SA. Protein disulfide isomerase was upregulated by application of either JA or SA. Proteins related to defense, energy, and protein destination/storage are suspected to be responsible for the susceptibility or resistance of the cultivars. Furthermore, in Roma, iron ABC transporter was upregulated by JA and downregulated by SA. Iron ABC transporter plays a part in the signal transduction of both JA and SA in cultivars of tomato that are resistant to bacterial wilt.

A novel class of L-lysine derivatives as aminopeptidase N (APN) inhibitors was designed and synthesized. Activity evaluation showed that compound C7 (IC50 = 9.6 ± 1.3 μM) and C20 (IC50 = 13.6 ± 1.9 μM) were equivalent to the positive control Bestatin (IC50 = 11.3 ± 1.6 μM).

Studies have shown that inverse proteins are relatively abundant. In this work, we investigate the proposition that the repeat patterns they share with protein sequences explain this phenomenon. Using a new artificial set of peptide sequences which also display these features and a random set, we show that the presence of repeats contributes to protein sequence similarity. Further analysis confirms that most inverse proteins exhibit repeats. Therefore, we suggest the relative abundance of inverse proteins can be explained by the fact they display the same repeat structures and amino acid propensity of existing proteins.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family which couple ATP hydrolysis with the transport of molecules across biological membranes. MRP6 topology presents three transmembrane domains and two nucleotide-binding domains (NBDs). The protein is structurally and functionally poorly characterized. Mutations in ABCC6 gene cause Pseudoxanthoma elasticum, a recessive genetic disorder affecting the elastic tissues. Most mutations have been found in NBDs that are critical for ATP binding and hydrolysis. With the aim to better characterize MRP6, we have performed a preliminary study on the fragment E748-A785 of MRP6- NBD1, with the wild type sequence and the R765Q mutation found in PXE affected patients. CD and NMR spectroscopy show the presence of helical structures in both peptides. Fluorescence experiments demonstrate that peptides bind ATP. The NMR structure of the mutated peptide is compared with the corresponding region of the MRP6-NBD1 modeled structure using as a template the X-ray structure of MRP1-NBD1. The finding that both wild type and mutated peptide present the same structure and similar affinity for ATP suggests that the onset of PXE symptoms is a consequence of the different type of interactions involving residue 765 R/Q inside the protein.

Two-component systems (TCS) involving the His-Asp phosphotransfer are commonly utilized for signal transduction in prokaryotes in which the two essential components are a sensor histidine kinase (HK) receptor and a response regulator (RR). Despite great efforts in structural and functional characterization of signal perception mechanisms, the exact signaling mechanisms remain elusive for many TCSs. Mimicking the natural TCS signaling pathways, chimeric receptor kinases and response regulators have been constructed through the process of swapping modular domains of related TCSs. To design chimeras with new signaling pathways, domains from different proteins that have little relationship at the primary structural level but carrying desirable functional properties can be conjoined to engineer novel TCSs. These chimeras maintain the ability to respond to environmental stimulants by regulating protein phosphorylation to produce downstream output signals. Depending on the nature of external signals, chimeric TCSs can serve as a novel tool not only to examine the natural signaling mechanisms in TCSs, but also for industrial and clinical applications.

The in vitro insulin unfolding had been studied using the “equilibrium unfolding” method where protein is unfolded by reducing reagents in the presence of trace amounts of oxidants such as oxidized glutathione. Nine intermediates were captured in the unfolding process, named as P1A, P2A, P3A, P4A, P3B, P4B, P5B, P6B, and P7B, which were all either A chain derivatives or B chain derivatives. No intermediate with inter-A-B chain disulfide was captured. Based on the character of the intermediates, their distribution during the unfolding process and the hypothetic “transient” intermediates, an in vitro putative unfolding pathway of insulin had been proposed. Besides, the comparison of the intermediates captured in unfolding with the intermediates captured in the refolding process of insulin revealed that both unfolding/ refolding processes of insulin shared common intermediates. Based on these observations we suggested that the unfolding pathway of insulin was similar to the refolding pathway but flowed in the opposite direction.

Taking all the proteins of four virus genomes as samples, the segments of α-helix and β-strand in proteins of the four viruses were obtained. Linear regression analyses between the average polarities and the folding rates of peptide chains were performed for α-helices and β-strands respectively. The results indicated that the folding rates show significant positive linear correlation for α-helices and negative linear correlation for β-strands with the average polarities. Based on the corresponding protein coding sequences of these amino acid segments, the influences of GC content of palindromes and palindrome densities in protein coding segments on the relations between the folding rates and the average polarities were studied. Results showed that the folding rates correlated positively with the GC content of palindromes and the palindrome density, and protein coding sequences do carry the information which can influence the folding rates of peptide chains or protein structures. Our analysis indicates that this kind of effect mostly comes from the information of palindrome structure itself or from the synonymous codon usage, but not from the translation information from codons to amino acids.

Bz(NO2)-Orn(Boc)-OCH2CN was synthesized as an amino acid component with effective and successful orthogonal protection for amino acylation of 5'-O-Pivaloyl nucleosides and preparation of substrates for model ribosome reactions. The synthesis was carried out using suitable combinations of the methods of peptide synthesis and modification of amino acids.

The transcription factor (TF) is a protein that binds DNA at specific site to help regulate the transcription from DNA to RNA. The mechanism of transcriptional regulatory can be much better understood if the category of transcription factors is known. We introduce a system which can automatically categorize transcription factors using their primary structures. A feature analysis strategy called “mRMR” (Minimum Redundancy, Maximum Relevance) is used to analyze the contribution of the TF properties towards the TF classification. mRMR is coupled with forward feature selection to choose an optimized feature subset for the classification. TF properties are composed of the amino acid composition and the physiochemical characters of the proteins. These properties will generate over a hundred features/parameters. We put all the features/parameters into a classifier, called NNA (nearest neighbor algorithm), for the classification. The classification accuracy is 93.81%, evaluated by a Jackknife test. Feature analysis using mRMR algorithm shows that secondary structure, amino acid composition and hydrophobicity are the most relevant features for classification. A free online classifier is available at http://app3.biosino.org/132dvc/tf/.

Human histatins are histidine-rich, low molecular weight salivary proteins that contribute to the immune system of the oral cavity. In this work, nucleotide sequences of the HIS1 (coding for histatin 1) and HIS2 (coding for histatin 3) genes, homologous to the human ones, have been sequenced and analysed in five primates species including Great Ape, Hylobatidae and Cercopithecidae. In HIS1, the region corresponding to the putative mature peptide shows a premature stop codon in Macaca and Cercopithecus, while HIS2 a six codon insertion in the Cercopithecidae. Histatin 5, a 24- residue peptide derived from histatin 3, is the most antimicrobially active among human histatins, thus macaque and nomascus orthologues of histatin 5 were selected for chemical synthesis and functional characterization, in comparison to the human peptide. All synthesized histatins are predicted to be poorly amphipathic, depending on the charged state of His residues and assume partially α-helical conformations only in lipophilic conditions. Antimicrobial assays against Candida and Criptococcus spp. indicate somewhat different spectra of in vitro activity against the tested fungi. We have described HIS1 and HIS2 gene variations in primates and have analysed their functional effects on selected Hst5 orthologues. The human antimicrobial peptide has been proposed to represent an important lead for new generation of antimicrobial compounds for the treatment of oral mycoses, thus the information from the non-human primates histatins studied may aid strategies for drugs design.

Overactivity of platelet-derived growth factor (PDGF) has been linked to malignant cancers. High levels of PDGF result in the activation of its receptors (PDGFRs) and the over-proliferation of cells. Therefore, interfering with this signaling pathway in cancer cells could be significant for anti-cancer drug development. In a previous study, the sPDGFRα-Fc fusion protein expressed in static CHO-k1 cells showed an anti-proliferative effect on vascular endothelial cells. However, it was difficult to obtain a large quantity of this fusion protein for further functional studies. In the present study, the sPDGFRα-Fc fusion protein was transiently expressed in Chinese Hamster Ovary (CHO) DG44 cells in 50-mL orbital shaking bioreactors. sPDGFRα-Fc was expressed as a 250-kDa dimeric protein with potential glycosylation. The final yield of sPDGFRα-Fc in the culture supernatant was as high as 16.68 mg/L. Our results suggest that transient expression in orbital shaking bioreactors may be feasible for preparation of recombinant proteins used for preclinical studies.

It has been recently reported that thiol groups could play an important role in the protection of neuronal cells in Alzheimer's disease (AD), prion disease (CJD) and Parkinson's disease (PD). Also bucillamine, that is a pseudo dipeptide possessing a thiol group capable to form an internal disulfide bridge, has relevant scavenger properties used in therapy for the treatment of arthritis. Furthermore, many sulphur containing compounds show strong chelating properties to heavy metals. Due to the crucial role of thiol groups in a variety of detoxicant biological systems, we report the synthesis of a racemic β,β-dialkyl-substituted, fully protected, cysteine derivative as a suitable intermediate in the synthesis of novel biological active peptides.