Specific Amino Acid Sequences Research Articles

Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions

Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.

Role of RecQ5 in Syncytial Blastoderm of the <i>Drosophila</i> Embryo

RecQ5 belongs to the family of RecQ DNA helicases. There are 5 RecQ homologs in mammals, and defects in 3 of them (BLM, WRN, and RECQL4) give rise to cancer predisposition syndromes in humans. Although no human disease has yet been genetically linked to a mutation in RecQ5, this enzyme is thought to have unique functions, based on its ubiquitous expression profile and specific C-terminal amino acid sequence, both of which are very different from those of other RecQ DNA helicase family members. The analysis of MEF and ES cells derived from RecQ5-deficient mice investigated by Hu et al. suggested an important role for RecQ5 in the DNA metabolism of the early embryo. However, it is unknown how RecQ5 deficiency destabilizes DNA. To address the DNA instabilities in RecQ5-deficient animals, we chose Drosophila melanogaster which has simple checkpoint systems in its syncytial embryos. By analyzing Drosophila syncytial embryos, we demonstrated that the loss of RecQ5 increased the frequency of spontaneous mitotic defects such as anaphase bridge formation. A pair of daughter nuclei that had been linked by such DNA bridges was simultaneously eliminated via a Chk2-dependent pathway. These findings suggest that the lack of RecQ5 causes spontaneous double-strand DNA breaks. RecQ5 may thus function in the resolution of anaphase DNA bridges during mitosis in syncytial embryo.

Amyloid beta–heme peroxidase promoted protein nitrotyrosination: relevance to widespread protein nitration in Alzheimer’s disease

Amyloid beta (Aβ) peptide accumulation has been demonstrated to play a central role in Alzheimer's disease (AD). Substantial evidence indicates that protein nitrotyrosination contributes to Aβ-dependent neurotoxicity; however, the molecular mechanism is unknown. Recent research has shown that Aβ complexes with heme to form Aβ-heme, and increases the pseudo-peroxidase activity of heme. We found that Aβ-heme uses H(2)O(2) and NO(2)(-) to cause nitration of enolase and synaptic proteins more effectively than heme. Thus, the increased peroxidase activity of Aβ-heme may be the molecular link between excess Aβ and the widespread protein nitration in AD. Interestingly, the site of enolase nitration that was catalyzed by Aβ-heme is different from that induced by heme. Moreover, the secondary structural perturbations of Aβ-heme-treated and heme-treated enolase are also different. These observations suggest that Aβ-heme targets specific amino acid sequences in enolase. Furthermore, our data show that Aβ-heme peroxidase activity is independent of the aggregation state of Aβ, suggesting an important role of soluble Aβ in addition to Aβ aggregates and oligomers in AD pathogenesis.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Electrochemical investigations of sarcoma-related protein kinase inhibition

An electrochemical biosensor was developed for the determination of sarcoma (Src)-related protein kinase-catalyzed phosphorylation reactions in the presence of adenosine 5′-γ-ferrocenoyl triphosphate (Fc-ATP). The sensing platform is based on a highly specific amino acid sequence Glu-Gly-Ile-Tyr-Asp-Val-Pro (EGIYDVP), to which a Fc-PO 2 moiety can be transferred from Fc-ATP by the action of the Src kinase. The enzyme kinetics and kinase inhibition were investigated by square wave voltammetry (SWV). The kinetic parameters K m and V max were determined for Src protein kinase with respect to Fc-ATP co-substrate and were found to be 200 μM and 115 μA cm −2 min, for phosphorylation of the EGIYDVP peptide substrate. Furthermore, the Src-catalyzed phosphorylation of Tyr was investigated in the presence of the small molecule inhibitors PP1, PP2, SU6656, and roscovitine. PP3 does not inhibit Src activity and was used as a control. The percent inhibition at half concentration, IC 50, values were determined for all inhibitors under the study and were estimated to be in the 5–30 nM range. The electrochemical study suggests that the increase in inhibition efficiency was in the order PP3 < SU6656 < roscovitine < PP2 < PP1.

Abstract 5268: CXCR4 and P-glycoprotein N-glycan patterning in developing cancer cells

Abstract Human CXCR4 and P-glycoprotein (Pgp) are N-glycosylated membrane proteins overexpressed in cancer cells. CXCR4 is thought to be an initiator of neuroblastoma metastasis to bone tissue and is overexpressed in neuroblastomas. Pgp is a heavily glycosylated, ATP-dependent drug efflux pump overexpressed in uterine sarcomas. Pgp is responsible for multidrug resistance in some tumors. Our lab is studying the N-glycan profiles of CXCR4 and Pgp in neuroblastoma (IMR-32) and uterine sarcoma cells (MES-SA and MES-SA/Dx5). We believe CXCR4 and Pgp are closely involved in metastasis. To better understand the molecular mechanisms of the metastatic roles of CXCR4 and Pgp, we are characterizing their respective complements of N-glycans. CXCR4 contains a N-linked oligosaccharide on two separate extracellular loops; moreover, Pgp has 3 N-glycans present on a 9 amino acid stretch near its N-terminus. We believe both protein N-glycan moieties are differentially glycosylated enough to act as markers for identifying and staging cancer cells. Our previous studies have shown that Pgp overexpressed in MES-SA/Dx5 cells contains at least two different complex N-glycans; a high mannose tree and a hybrid, sialic acid-capped oligosaccharide. To build on our previous studies, we have developed a novel approach to further characterize CXCR4 and Pgp N-glycans using polyclonal antibodies (pAbs) raised to the specific amino acid sequences containing the sugars in both cancer proteins. We are using these inimitable pAbs to aid in the isolation, sequencing, and profiling of specific N-glycan changes that are associated with the stage of each of the cancer cell lines mentioned above. Our pAb panels bind the native, expressed glycoforms of CXCR4 and Pgp in cells and on Western blots. We are using pAb affinity columns to capture protease-released glycopeptides. Once captured, we intend to sequence each of the released glycopeptides’ oligosaccharides to verify their saccharide sequences in the different cancer cell stages. Furthermore, we predict that the resultant glycopeptide gel and blot patterns will ‘bar code’ the specific cancer stage of the cells lines studied. We envision that these glycopeptide patterns can be used to identify and catalog the various stages of developing cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2011-5268

Domain Movement within a Gene: A Novel Evolutionary Mechanism for Protein Diversification

A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.

Classification of viral zoonosis through receptor pattern analysis

BackgroundViral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses). Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell.ResultsWe analysed host-cell receptor sequences for their hydrophobicity/hydrophilicity characteristics. We then analysed these properties for similarities among receptors of different species and used a statistical discriminant analysis to predict the likelihood of transmission among species.ConclusionsThis study is an attempt to predict zoonosis through simple computational analysis of receptor sequence differences. Our method may be useful in predicting the zoonotic potential of newly discovered viral strains.

MHC variation, multiple simultaneous infections and physiological condition in the subterranean rodent Ctenomys talarum

Parasites and pathogens can play a significant role in shaping the genetic diversity of host populations, particularly at genes associated with host immune response. To explore this relationship in a natural population of vertebrates, we characterized Major Histocompatibility Complex (MHC) variation in the subterranean rodent Ctenomys talarum (the talas tuco-tuco) as a function of parasite load and ability to mount an adaptive immune response against a novel antigen. Specifically, we quantified genotypic diversity at the MHC class II DRB locus in relation to (1) natural variation in infection by multiple genera of parasites (potential agents of selection on MHC genes) and (2) antibody production in response to injection with sheep red blood cells (a measure of immunocompetence). Data were analyzed using co-inertia multivariate statistics, with epidemiological proxies for individual condition (hematocrit, leukocyte profile, body weight) and risk of parasite exposure (season of capture, sex). A significant excess of DRB heterozygotes was evident in the study population. Co-inertia analyses revealed significant associations between specific DRB alleles and both parasite load and intensity of humoral immune response against sheep red blood cells. The presence of specific DRB aminoacid sequences appeared to be more strongly associated with parasite load and response to a novel antigen than was heterozygosity at the DRB locus. These data suggest a role for parasite-driven balancing selection in maintaining MHC variation in natural populations of C. talarum. At the same time, these findings underscore the importance of using diverse parameters to study interactions among physiological conditions, immunocompetence, and MHC diversity in free-living animals that are confronted with multiple simultaneous immune challenges.

P85α Regulates Osteoblast Differentiation by Cross-talking with the MAPK Pathway

Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.

Development of a sandwich enzyme linked immunosorbent assay (ELISA) for the quantification of iduronate-2-sulfate sulfatase

Iduronate-2-sulfate sulfatase (IDS; EC 3.1.6.13) is an enzyme that belongs to human sulfatases. IDS deficiency causes the Hunter syndrome or mucopolysaccharidosis type II (MPS II; OMIM 309900). We have been developing an expression system for human recombinant IDS (hrIDS) in Pichia pastoris, therefore a method was required for its detection during production and purification processes, which could be used also to measure the enzyme in human fluids. In this study, an immunoquantification assay for human and recombinant IDS was developed with the combination of two antibodies. Rabbit IgG and chicken IgY were used as IDS capture and detection antibodies, respectively. Chicken IgY antibodies were developed against specific amino acid sequences present in IDS but absent in other human sulfatases. hrIDS produced in P. pastoris, commercial hrIDS, and normal human plasma samples were used as antigens and immunoquantification results were compared to enzyme activity. The technique was linear over the range 8 to 500ngmL−1 using commercial hrIDS. The concentration range detected for IDS in normal human plasma was 14.43 to 287.88ngmL−1. The hrIDS was detected in P. pastoris cultures even when the enzyme was inactive, which is convenient for monitoring the production of recombinant proteins. These results show that chicken site-specific antibodies provide a good alternative, as a substitute of monoclonal antibodies, for the detection of human proteins. This is the first report on the development of an ELISA system to detect and quantify IDS with IgY antibodies.

Identification of Target Ligands of CORYNE in Arabidopsis by Phage Display Library

CORYNE (CRN) plays important roles in stem cell division and differentiation of shoot apical meristem (SAM) in Arabidopsis thaliana. The cytoplasmic kinase domain of CRN has been cloned and expressed in Escherichia coli, and further purified by two consecutive steps of affinity chromatography. By using this purified CRN as a ligand, a 12-mer random-peptide library was used to determine the specific amino acid sequences binding with the recombinant CRN molecule. After four rounds of biopanning, positive phage clones were isolated and sequenced, and further tested by enzyme linked immunosorbent assay for their binding ability and specificity. Two positive clones that specifically bind to the intracellular protein kinase domain of CRN have been identified. Alignment of these peptides and the kinase-associated protein phosphatase (KAPP) shows high similarity, indicating that KAPP might interact with the cytoplasmic kinase domain of CRN and negatively regulate the CLV signal. Our current study would be helpful to better understand the CLV3 signal pathway.

Species specific amino acid sequence–protein local structure relationships: An analysis in the light of a structural alphabet

Protein structure analysis and prediction methods are based on non-redundant data extracted from the available protein structures, regardless of the species from which the protein originates. Hence, these datasets represent the global knowledge on protein folds, which constitutes a generic distribution of amino acid sequence–protein structure (AAS–PS) relationships. In this study, we try to elucidate whether the AAS–PS relationship could possess specificities depending on the specie. For this purpose, we have chosen three different species: Saccharomyces cerevisiae, Plasmodium falciparum and Arabidopsis thaliana. We analyzed the AAS–PS behaviors of the proteins from these three species and compared it to the “expected” distribution of a classical non-redundant databank. With the classical secondary structure description, only slight differences in amino acid preferences could be observed. With a more precise description of local protein structures (Protein Blocks), significant changes could be highlighted. S. cerevisiae's AAS–PS relationship is close to the general distribution, while striking differences are observed in the case of A. thaliana. P. falciparum is the most distant one. This study presents some interesting view-points on AAS–PS relationship. Certain species exhibit unique preferences for amino acids to be associated with protein local structural elements. Thus, AAS–PS relationships are species dependent. These results can give useful insights for improving prediction methodologies which take the species specific information into account.

Na + , K + -ATPase

The adenosine triphosphatases (ATPases) are integral membrane proteins that actively exchange ions across cell membranes; the energy required for this process is derived from hydrolysis of one molecule of ATP. The sodium (Na+), potassium (K+) ATPase (Na+, K+-ATPase) simultaneously extrudes 3 Na+ ions from the intracellular compartment in exchange for 2 K+ ions from the extracellular space. The Na+, K+-ATPase maintains the Na+ and K+ gradients that are of fundamental importance for maintenance of neuronal excitability and conduction of the action potential and for secondary transport systems involved in synaptic uptake of neurotransmitters and regulation of cell volume, pH, and calcium (Ca2+) concentrations. The Na+, K+-ATPase forms macromolecular complexes with other membrane proteins and triggers intracellular transduction signals involved in synaptic plasticity. There are different isoforms of the Na+, K+-ATPase expressed in neurons and glial cells. Impaired Na+, K+-ATPase activity, resulting from reduced ATP availability due to mitochondrial dysfunction in the setting of hypoxia, ischemia, inflammation, and other conditions, triggers neuronal depolarization, cytotoxic edema, and neuronal, axonal, and glial injury. The elucidation of the cristal structure of the catalytic α subunit of the Na+, K+-ATPase and the functional consequences of specific mutations have led to identification of specific amino acid sequences that are critical for the normal function of the enzyme. Mutations affecting the α2 subunit of the Na+, K+-ATPase, which is expressed in astrocytes, is linked to familial hemiplegic migraine type 2 (FHM2); mutations affecting the α3 subunit, which is expressed in neurons, are linked to rapid onset dystonia and parkinsonism. The structure and function of the Na+, K+-ATPase and …

Molecular and structural mapping of collagen fibril interactions

The fibrous collagens form the structural basis of all mammalian connective tissues, including the vasculature, dermis, bones, tendons, cartilage, and those tissues that support organs such as the heart, kindneys, liver, and lungs. The helical structure of collagen has been extensively studied but in addition to its helical character, its molecular packing arrangement (in its aggregated or fibrillar form) and the presence of specific amino acid sequences govern collagen's in vivo functions. Collagen's molecular packing arrangement helps control cellular communication, attachment and movement, and conveys its tissue-specific biomechanical properties. Recent progress in understanding collagen's molecular packing, fibrillar structure, domain organization, and extracellular matrix (ECM) interactions in light of X-ray fiber diffraction data provides significant new insights into how the ECM is organized and functions. In this review, the hierarchy of fibrillar collagen structure is discussed in the context of how this organization affects ECM–“ligand” interactions, with specific attention to collagenolysis, integrins, fibronection, glycoprotein VI receptor (GPVI), and proteoglycans (PG). Understanding the complex structure of collagen and its attached ligands should provide new insights into tissue growth, development, regeneration, and disease.

Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aβ oligomers

BackgroundAge-related neurodegenerative diseases share a number of important pathological features, such as accumulation of misfolded proteins as amyloid oligomers and fibrils. Recent evidence suggests that soluble amyloid oligomers and not the insoluble amyloid fibrils may represent the primary pathological species of protein aggregates.ResultsWe have produced several monoclonal antibodies that specifically recognize prefibrillar oligomers and do not recognize amyloid fibrils, monomer or natively folded proteins. Like the polyclonal antisera, the individual monoclonals recognize generic epitopes that do not depend on a specific linear amino acid sequence, but they display distinct preferences for different subsets of prefibrillar oligomers. Immunological analysis of a number of different prefibrillar Aβ oligomer preparations show that structural polymorphisms exist in Aβ prefibrillar oligomers that can be distinguished on the basis of their reactivity with monoclonal antibodies. Western blot analysis demonstrates that the conformers defined by the monoclonal antibodies have distinct size distributions, indicating that oligomer structure varies with size. The different conformational types of Aβ prefibrillar oligomers can serve as they serve as templates for monomer addition, indicating that they seed the conversion of Aβ monomer into more prefibrillar oligomers of the same type.ConclusionsThese results indicate that distinct structural variants or conformers of prefibrillar Aβ oligomers exist that are capable of seeding their own replication. These conformers may be analogous to different strains of prions.

Mass Spectrometric Characterization of Modifications to Angiotensin II by Lipid Peroxidation Products, 4-Oxo-2(E)-nonenal and 4-Hydroxy-2(E)-nonenal

The octapeptide angiotensin II (Ang II; Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)) is the primary active hormone of the renin/angiotensin system (RAS) and has been implicated in various cardiovascular diseases. Numerous structure-activity relationship studies have identified Asp(1), Arg(2), and His(6) of Ang II to be critical for its biological activity and receptor binding. From the reactions of Ang II with lipid peroxidation-derived aldehydes, 4-oxo-2(E)-nonenal (ONE) or 4-hydroxy-2(E)-nonenal (HNE), we have identified the major modifications to the N-terminus, Asp(1), Arg(2), and His(6) of Ang II by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption ionization-time-of-flight/MS (MALDI-TOF/MS). The identities of ONE- and HNE-modified Ang II were confirmed by tandem mass spectrometry (MS/MS) and postsource decay (PSD)-TOF/MS before and after the reaction with sodium borohydride. In the reaction with ONE, a pyruvamide-Ang II that formed via oxidative decarboxylation of N-terminal Asp was detected as the most abundant product after 48 h of incubation. It was followed by Arg-modified [Arg(2)(ONE-H(2)O)]-Ang II and the N-terminal-modified 4-ketoamide form of [N-ONE]-Ang II. The Michael addition products of [His(6)(HNE)]-Ang II were the most abundant products in the beginning of the reaction with HNE, followed by the dehydrated Michael addition products of [His(6)(HNE-H(2)O)]-Ang II. [His(6)(HNE)]-Ang II was dehydrated to [His(6)(HNE-H(2)O)]-Ang II during the prolonged incubation, and [His(6)(HNE-H(2)O)]-Ang II became the major products after 7 days. The model reactions of N(α)-tert-butoxycarbonyl (tBoc)-Arg with ONE and tBoc-His with HNE were performed and compared with the Ang II reaction. tBoc-Arg readily reacted with ONE to produce a compound analogous to [Arg(2)(ONE-H(2)O)]-Ang II, which confirmed Arg as one of the important target nucleophiles of ONE. However, tBoc-His exclusively formed a Michael addition product upon the reaction with HNE. The unexpected formation of [His(6)(HNE-H(2)O)]-Ang II can be explained by the proximity of His(6) to C-terminal carboxylate in the specific conformation of Ang II, which facilitates the dehydration of Michael addition products. Therefore, our results suggest a possible discrepancy in the adduction chemistry of ONE and HNE for model amino acids and endogenous bioactive peptides, which is governed by the microenvironment of peptides, such as the specific amino acid sequence and conformation. Such stable ONE- and HNE-derived modifications to Ang II could potentially modulate its functions in vivo by disrupting the interaction with Ang II type 1 (AT(1)) receptor and/or inhibiting the enzyme activity of aminopeptidase A (APA), which cleaves the N-terminal Asp residue of Ang II to generate Ang III.

Fold versus sequence effects on the driving force for protein‐mediated electron transfer

Electron transport chains composed of electron transfer reactions mainly between proteins provide fast efficient flow of energy in a variety of metabolic pathways. Reduction potentials are essential characteristics of the proteins because they determine the driving forces for the electron transfers. As both polar and charged groups from the backbone and side chains define the electrostatic environment, both the fold and the sequence will contribute. However, although the role of a specific sequence may be determined by experimental mutagenesis studies of reduction potentials, understanding the role of the fold by experiment is much more difficult. Here, continuum electrostatics and density functional theory calculations are used to analyze reduction potentials in [4Fe-4S] proteins. A key feature is that multiple homologous proteins in three different folds are compared: six high potential iron-sulfur proteins, four bacterial ferredoxins, and four nitrogenase iron proteins. Calculated absolute reduction potentials are shown to be in quantitative agreement with electrochemical reduction potentials. Calculations further demonstrate that the contribution of the backbone is larger than that of the side chains and is consistent for homologous proteins but differs for nonhomologous proteins, indicating that the fold is the major protein factor determining the reduction potential, whereas the specific amino acid sequence tunes the reduction potential for a given fold. Moreover, the fold contribution is determined mainly by the proximity of the redox site to the protein surface and the orientation of the dipoles of backbone near the redox site.

Cationic Amphiphiles, a New Generation of Antimicrobials Inspired by the Natural Antimicrobial Peptide Scaffold

Naturally occurring cationic antimicrobial peptides (AMPs) and their mimics form a diverse class of antibacterial agents currently validated in preclinical and clinical settings for the treatment of infections caused by antimicrobial-resistant bacteria. Numerous studies with linear, cyclic, and diastereomeric AMPs have strongly supported the hypothesis that their physicochemical properties, rather than any specific amino acid sequence, are responsible for their microbiological activities. It is generally believed that the amphiphilic topology is essential for insertion into and disruption of the cytoplasmic membrane. In particular, the ability to rapidly kill bacteria and the relative difficulty with which bacteria develop resistance make AMPs and their mimics attractive targets for drug development. However, the therapeutic use of naturally occurring AMPs is hampered by the high manufacturing costs, poor pharmacokinetic properties, and low bacteriological efficacy in animal models. In order to overcome these problems, a variety of novel and structurally diverse cationic amphiphiles that mimic the amphiphilic topology of AMPs have recently appeared. Many of these compounds exhibit superior pharmacokinetic properties and reduced in vitro toxicity while retaining potent antibacterial activity against resistant and nonresistant bacteria. In summary, cationic amphiphiles promise to provide a new and rich source of diverse antibacterial lead structures in the years to come.

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Ulva pertusa (Ulvophyceae, Chlorophyta)

In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone a full-length cytosolic heat shock protein 70 of Ulva pertusa (designated as UPHsp70). Bioinformatics was used to analyze structural features, homologous relationship, and phylogenetic position of UPHsp70. The full length of UPHsp70 cDNA was 2,283 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 247 bp, and an open reading frame of 1,971 bp encoding a polypeptide of 656 amino acids with an estimated molecular weight of 71.13 kDa and an estimated isoelectric point of 5.04. The UPHsp70 had four degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The specific C-terminus amino acid sequence of cytosolic UPHsp70 was EEVD, and the conservation of Hsp70s of N-terminus was higher than that of C-terminus. The homology between UPHsp70 and Hsp70s of other known algae and land plants was more than 70%. Under different stress conditions, mRNA expression levels of UPHsp70 were quantified by quantitative reverse transcriptase-polymerase chain reaction. When U. pertusa samples were kept in different temperatures (5–40°C) for 1 h, the expression level of UPHsp70 at 5°C, 35°C, or 40°C was over onefold higher than that at 25°C. When U. pertusa samples were kept at 30°C for different times (0–12 h), the mRNA expression level of UPHsp70 had a trend of rise first then fall. The expression level of UPHsp70 reached maximum level after 5 h. When U. pertusa samples were kept in different salt concentrations (0–45‰) for 2 h, the expression level of UPHsp70 at 0‰ or 5‰ salt concentration was twofold higher than that at 30‰ for 2 h. The expression levels of UPHsp70 at 30‰, 35‰, and 40‰ were low and had no difference (P < 0.05). When U. pertusa samples were kept at ultraviolet irradiation or desiccated for different times (0–4 h), the mRNA expression level of UPHsp70 reached maximum level after 3.0 h; after that, it was maintained at high level.