Showing papers on "Peptide sequence published in 2012"
TL;DR: The γ-secretase complex and its cell surface localization, in the absence of an effect on Notch, is studied to avoid side-effects caused by EP2 receptor.
Abstract: IW Hamley Chemical reviews, 2012 ACS Publications ... the γ-secretase complex and its cell surface localization, in the absence of an effect on Notch ... in the development of effective γ-secretase inhibitors is to avoid side-effects caused by ... EP2 receptor.(79) Prostaglandin E 2 is produced during inflammation due to activity by cytosolic ... Cited by 143 Related articles All 4 versions Cite Save
735 citations
TL;DR: It is shown that leucyl-tRNA synthetase (LRS) plays a critical role in amino acid-induced mTORC1 activation by sensing intracellular leucine concentration and initiating molecular events leading to mTORc1 activation.
706 citations
TL;DR: It is shown that amino acid covariation in proteins, extracted from the evolutionary sequence record, can be used to fold transmembrane proteins, and how the method can predict oligomerization, functional sites, and conformational changes in transmemBRane proteins is shown.
529 citations
TL;DR: These findings provide the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.
Abstract: Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 A resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS8–13, the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity. The X-ray crystal structure of a rat neurotensin receptor in complex with the C-terminal portion of neurotensin is presented; this is the first structure of a member of the β group of class A G-protein-coupled receptors. Neurotensin is a short peptide that can act as a neurotransmitter, a digestive hormone, and a regulator of cardiac output and blood pressure. In this manuscript, the authors solve an X-ray crystal structure of the carboxy-terminal portion of neurotensin bound to a rat neurotensin receptor. This is the first structure of a member of the beta group of class A G-protein-coupled receptors (GPCRs), and the first published structure of a GPCR bound to a peptide agonist. This structure should facilitate the development of non-peptide drugs that could be used to treat neurological disorders, cancer and obesity.
466 citations
TL;DR: A high-throughput quantitative method is developed for a comprehensive single-mutation study in which every position is substituted individually to every other amino acid and shows that sector positions are functionally sensitive to mutation, whereas non-sector positions are more tolerant to substitution.
Abstract: Statistical analysis of protein evolution suggests a design for natural proteins in which sparse networks of coevolving amino acids (termed sectors) comprise the essence of three-dimensional structure and function1, 2, 3, 4, 5 However, proteins are also subject to pressures deriving from the dynamics of the evolutionary process itself—the ability to tolerate mutation and to be adaptive to changing selection pressures6, 7, 8, 9, 10 To understand the relationship of the sector architecture to these properties, we developed a high-throughput quantitative method for a comprehensive single-mutation study in which every position is substituted individually to every other amino acid Using a PDZ domain (PSD95pdz3) model system, we show that sector positions are functionally sensitive to mutation, whereas non-sector positions are more tolerant to substitution In addition, we find that adaptation to a new binding specificity initiates exclusively through variation within sector residues A combination of just two sector mutations located near and away from the ligand-binding site suffices to switch the binding specificity of PSD95pdz3 quantitatively towards a class-switching ligand The localization of functional constraint and adaptive variation within the sector has important implications for understanding and engineering proteins
451 citations
TL;DR: NMR titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis and may aid in the design of Alzheimer’s therapeutics.
Abstract: C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.
438 citations
TL;DR: Phylogenetic analysis of gene families at the organism and sequence level revealed complex relationships between the evolution of this protein superfamily sequence and the acquisition of distinct cellular functions.
Abstract: The Ras superfamily is a fascinating example of functional diversification in the context of a preserved structural framework and a prototypic GTP binding site. Thanks to the availability of complete genome sequences of species representing important evolutionary branch points, we have analyzed the composition and organization of this superfamily at a greater level than was previously possible. Phylogenetic analysis of gene families at the organism and sequence level revealed complex relationships between the evolution of this protein superfamily sequence and the acquisition of distinct cellular functions. Together with advances in computational methods and structural studies, the sequence information has helped to identify features important for the recognition of molecular partners and the functional specialization of different members of the Ras superfamily.
372 citations
TL;DR: The structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis is established and its functional relevance in the regulation of NRF2 by the signaling pathways that impinge on G SKS-3 is established.
Abstract: The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.
367 citations
TL;DR: It is concluded that there are general shared features of bioactive peptides across different functional classes, indicating that computational prediction may accelerate the discovery of novel bio active peptides and aid in the improved design of existing peptides, across many functional classes.
Abstract: The conventional wisdom is that certain classes of bioactive peptides have specific structural features that endow their particular functions. Accordingly, predictions of bioactivity have focused on particular subgroups, such as antimicrobial peptides. We hypothesized that bioactive peptides may share more general features, and assessed this by contrasting the predictive power of existing antimicrobial predictors as well as a novel general predictor, PeptideRanker, across different classes of peptides.
We observed that existing antimicrobial predictors had reasonable predictive power to identify peptides of certain other classes i.e. toxin and venom peptides. We trained two general predictors of peptide bioactivity, one focused on short peptides (4–20 amino acids) and one focused on long peptides ( amino acids). These general predictors had performance that was typically as good as, or better than, that of specific predictors. We noted some striking differences in the features of short peptide and long peptide predictions, in particular, high scoring short peptides favour phenylalanine. This is consistent with the hypothesis that short and long peptides have different functional constraints, perhaps reflecting the difficulty for typical short peptides in supporting independent tertiary structure.
We conclude that there are general shared features of bioactive peptides across different functional classes, indicating that computational prediction may accelerate the discovery of novel bioactive peptides and aid in the improved design of existing peptides, across many functional classes. An implementation of the predictive method, PeptideRanker, may be used to identify among a set of peptides those that may be more likely to be bioactive.
295 citations
TL;DR: This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure.
285 citations
TL;DR: It was found that CRGDK peptides increased intracellular uptake of AuNPs compared to other surface conjugations quantified by ICP-MS, which has implications to design and functionalize different molecules onto AuNBP surfaces to make hybrid model system for selective target binding as well as therapeutic effects for cancer treatment.
TL;DR: A ribosomal origin is demonstrated of the marine sponge–derived polytheonamides, exceptionally potent, giant natural-product toxins, which broadens the biosynthetic scope of ribosomally synthesized peptides and proteins and creates new opportunities for peptide and protein bioengineering.
Abstract: It is held as a paradigm that ribosomally synthesized peptides and proteins contain only l-amino acids. We demonstrate a ribosomal origin of the marine sponge–derived polytheonamides, exceptionally potent, giant natural-product toxins. Isolation of the biosynthetic genes from the sponge metagenome revealed a bacterial gene architecture. Only six candidate enzymes were identified for 48 posttranslational modifications, including 18 epimerizations and 17 methylations of nonactivated carbon centers. Three enzymes were functionally validated, which showed that a radical S-adenosylmethionine enzyme is responsible for the unidirectional epimerization of multiple and different amino acids. Collectively, these complex alterations create toxins that function as unimolecular minimalistic ion channels with near-femtomolar activity. This study broadens the biosynthetic scope of ribosomal systems and creates new opportunities for peptide and protein bioengineering.
TL;DR: Collision cross section values (Ω) in both He and N(2) gases for 113 peptide ions determined directly from drift times measured in a low-pressure, ambient temperature drift cell with radio-frequency (rf) ion confinement enable the rapid and accurate determination of calibrated Ω for peptides.
Abstract: One difficulty in using ion mobility (IM) mass spectrometry (MS) to improve the specificity of peptide ion assignments is that IM separations are performed using a range of pressures, gas compositions, temperatures, and modes of separation, which makes it challenging to rapidly extract accurate shape parameters. We report collision cross section values (Ω) in both He and N2 gases for 113 peptide ions determined directly from drift times measured in a low-pressure, ambient temperature drift cell with radio-frequency (rf) ion confinement. These peptide ions have masses ranging from 231 to 2969 Da, ΩHe of 89–616 A2, and ΩN2 of 151–801 A2; thus, they are ideal for calibrating results from proteomics experiments. These results were used to quantify the errors associated with traveling-wave Ω measurements of peptide ions and the errors concomitant with using drift times measured in N2 gas to estimate ΩHe. More broadly, these results enable the rapid and accurate determination of calibrated Ω for peptide ions, w...
TL;DR: This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O- GloverNAc site assignment, and support the proposed regulatory cross-talk between O- GlcNAcylation and phosphorylation.
Abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.
TL;DR: It is shown that the inclusion of an additional linker with a length of four residues (-PPPPC) and a rigid, hydrophobic nature is a better choice for forming peptide self-assembled monolayers (SAMs) with a well-ordered structure and high surface density.
Abstract: Cysteine is commonly used to attach peptides onto gold surfaces. Here we show that the inclusion of an additional linker with a length of four residues (-PPPPC) and a rigid, hydrophobic nature is a better choice for forming peptide self-assembled monolayers (SAMs) with a well-ordered structure and high surface density. We compared the structure and function of the nonfouling peptide EKEKEKE-PPPPC-Am with EKEKEKE-C-Am. Circular dichroism, attenuated total internal reflection Fourier transform IR spectroscopy, and molecular dynamics results showed that EKEKEKE-PPPPC-Am forms a secondary structure while EKEKEKE-C-Am has a random structure. Surface plasmon resonance sensor results showed that protein adsorption on EKEKEKE-PPPPC-Am/gold is very low with small variation while protein adsorption on EKEKEKE-C-Am/gold is high with large variation. X-ray photoelectron spectroscopy results showed that both peptides have strong gold-thiol binding with the gold surface, indicating that their difference in protein adsorption is due to their assembled structures. Further experimental and simulation studies were performed to show that -PPPPC is a better linker than -PC, -PPC, and -PPPC. Finally, we extended EKEKEKE-PPPPC-Am with the cell-binding sequence RGD and demonstrated control over specific versus nonspecific cell adhesion without using poly(ethylene glycol). Adding a functional peptide to the nonfouling EK sequence avoids complex chemistries that are used for its connection to synthetic materials.
TL;DR: Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissociation (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation.
Abstract: Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissociation (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissociation step, all ions including the unreacted precursor ions are subjected to collision induced dissociation which yields b/y- and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification.
TL;DR: SSE combines sequence editor functions with analytical tools in a comprehensive and user-friendly package that assists considerably in bioinformatic and evolution research.
Abstract: There is an increasing need to develop bioinformatic tools to organise and analyse the rapidly growing amount of nucleotide and amino acid sequence data in organisms ranging from viruses to eukaryotes. A simple sequence editor (SSE) was developed to create an integrated environment where sequences can be aligned, annotated, classified and directly analysed by a number of built-in bioinformatic programs. SSE incorporates a sequence editor for the creation of sequence alignments, a process assisted by integrated CLUSTAL/MUSCLE alignment programs and automated removal of indels. Sequences can be fully annotated and classified into groups and annotated of sequences and sequence groups and access to analytical programs that analyse diversity, recombination and RNA secondary structure. Methods for analysing sequence diversity include measures of divergence and evolutionary distances, identity plots to detect regions of nucleotide or amino acid homology, reconstruction of sequence changes, mono-, di- and higher order nucleotide compositional biases and codon usage. Association Index calculations, GroupScans, Bootscanning and TreeOrder scans perform phylogenetic analyses that reconcile group membership with tree branching orders and provide powerful methods for examining segregation of alleles and detection of recombination events. Phylogeny changes across alignments and scoring of branching order differences between trees using the Robinson-Fould algorithm allow effective visualisation of the sites of recombination events. RNA secondary and tertiary structures play important roles in gene expression and RNA virus replication. For the latter, persistence of infection is additionally associated with pervasive RNA secondary structure throughout viral genomic RNA that modulates interactions with innate cell defences. SSE provides several programs to scan alignments for RNA secondary structure through folding energy thermodynamic calculations and phylogenetic methods (detection of co-variant changes, and structure conservation between divergent sequences). These analyses complement methods based on detection of sequence constraints, such as suppression of synonymous site variability. For each program, results can be plotted in real time during analysis through an integrated graphics package, providing publication quality graphs. Results can be also directed to tabulated datafiles for import into spreadsheet or database programs for further analysis. SSE combines sequence editor functions with analytical tools in a comprehensive and user-friendly package that assists considerably in bioinformatic and evolution research.
TL;DR: GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results.
Abstract: Background
Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task.
TL;DR: This ‘improved’ recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far, and site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.
Abstract: Uniquely among RNA viruses, replication of the ~30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.
TL;DR: The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface.
Abstract: Mdm2 is a major negative regulator of the tumor suppressor p53 protein, a protein that plays a crucial role in maintaining genome integrity. Inactivation of p53 is the most prevalent defect in human cancers. Inhibitors of the Mdm2–p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. We present here a 2.0 A resolution structure of the Mdm2 protein with a bound stapled p53 peptide. Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein–protein interactions and thus have broad therapeutic potential. The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface.
TL;DR: Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity.
Abstract: Background: Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B� 57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on 1) HLA-B� 57:01 epitope-binding in vitro and 2) the quality and quantity of self-peptides presented by HLA-B� 57:01 from abacavir-treated cells. Design and methods: An HLA-B� 57:01 specific epitope binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B� 57:01, a B-cell line secreting soluble HLA (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified HLA, and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Results: Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC labeled self-peptide LF9 to HLA-B� 57:01 in a dose dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B� 57:01 B-cells showed amino acid sequence differences compared with peptides from untreated cells. Novel druginduced (DI)-peptides lacked typical carboxyl(C)-terminal amino-acids characteristic of the HLA-B� 57:01 peptide motif and instead contained predominantly Isoleucine or Leucine residues. DI-peptides bind to soluble HLA-B� 57:01 with high affinity that was not altered by abacavir addition. Conclusion: Our results support a model of drug-induced autoimmunity in which abacaviraltersthequantity andquality of self-peptideloadinginto HLA-B� 57:01.Druginduced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide loading complex, generates an array of neo
TL;DR: The truncated form of LyP-1, in which the CendR motif is exposed, is shown to be a promising tool for targeted delivery of therapeutic and diagnostic agents to breast cancers and perhaps other types of tumors.
Abstract: We have recently described a class of peptides that improve drug delivery by increasing penetration of drugs into solid tumors These peptides contain a C-terminal C-end Rule (CendR) sequence motif (R/K)XX(R/K), which is responsible for cell internalization and tissue-penetration activity Tumor-specific CendR peptides contain both a tumor-homing motif and a cryptic CendR motif that is proteolytically unmasked in tumor tissue A previously described cyclic tumor-homing peptide, LyP-1 (sequence: CGNKRTRGC), contains a CendR element and is capable of tissue penetration We use here the truncated form of LyP-1, in which the CendR motif is exposed (CGNKRTR; tLyP-1), and show that both LyP-1 and tLyP-1 internalize into cells through the neuropilin-1-dependent CendR internalization pathway Moreover, we show that neuropilin-2 also binds tLyP-1 and that this binding equally activates the CendR pathway Fluorescein-labeled tLyP-1 peptide and tLyP-1-conjugated nanoparticles show robust and selective homing to tumors, penetrating from the blood vessels into the tumor parenchyma The truncated peptide is more potent in this regard than the parent peptide LyP-1 tLyP-1 furthermore improves extravasation of a co-injected nanoparticle into the tumor tissue These properties make tLyP-1 a promising tool for targeted delivery of therapeutic and diagnostic agents to breast cancers and perhaps other types of tumors
TL;DR: The combined YmdB-ClsC gene pair used PE as the phosphatidyl donor to PG to form CL, which demonstrates a third and unique mode for CL synthesis.
Abstract: Depending on growth phase and culture conditions, cardiolipin (CL) makes up 5–15% of the phospholipids in Escherichia coli with the remainder being primarily phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). In E. coli, the cls and ybhO genes (renamed clsA and clsB, respectively) each encode a CL synthase (Cls) that catalyzes the condensation of two PG molecules to form CL and glycerol. However, a ∆clsAB mutant still makes CL in the stationary phase, indicating the existence of additional Cls. We identified a third Cls encoded by ymdC (renamed clsC). ClsC has sequence homology with ClsA and ClsB, which all belong to the phospholipase D superfamily. The ∆clsABC mutant lacks detectible CL regardless of growth phase or growth conditions. CL can be restored to near wild-type levels in stationary phase in the triple mutant by expressing either clsA or clsB. Expression of clsC alone resulted in a low level of CL in the stationary phase, which increased to near wild-type levels by coexpression of its neighboring gene, ymdB. CL synthesis by all Cls is increased with increasing medium osmolarity during logarithmic growth and in stationary phase. However, only ClsA contributes detectible levels of CL at low osmolarity during logarithmic growth. Mutation of the putative catalytic motif of ClsC prevents CL formation. Unlike eukaryotic Cls (that use PG and CDP-diacylglycerol as substrates) or ClsA, the combined YmdB-ClsC used PE as the phosphatidyl donor to PG to form CL, which demonstrates a third and unique mode for CL synthesis.
TL;DR: A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain, and irreversible calpain family activity-based probes (ABPs) which retained the specificity of the stabilized helical inhibitor were developed.
Abstract: We have designed a highly specific inhibitor of calpain by mimicking a natural protein–protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), wh...
TL;DR: This chapter describes the structure and function of phage contractile tails and compares them with other phage tails and with other known contractile systems.
Abstract: Bacteriophages with contractile tails epitomize the concepts of "virus" and "phage" for many because the tails of these phages undergo a large conformational change - resembling the action of a syringe - upon the attachment to the host cell. The contractile tails belong to the recently recognized class of "contractile systems," which includes phage tails, their close relatives R-type pyocins, the bacterial type VI secretion system, and the virulence cassette of Photorhabdus. Their function is to deliver large proteins and/or DNA into the cytoplasm of a bacterial or eukaryotic cell. The structure of the core components of all contractile tail-like systems is conserved, but the corresponding genes have diverged to such a degree that the common ancestry can no longer be easily detected at the level of amino acid sequence. At present, it is unclear, whether the contractile systems originated in bacteria or in phages. This chapter describes the structure and function of phage contractile tails and compares them with other phage tails and with other known contractile systems.
United States Department of the Army1, Walter Reed Army Institute of Research2, New York University3, Veterans Health Administration4, National Institutes of Health5, Duke University6, Fred Hutchinson Cancer Research Center7, Thailand Ministry of Public Health8, Mahidol University9, Sanofi Pasteur10, Los Alamos National Laboratory11
TL;DR: Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
Abstract: The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved ep...
TL;DR: The results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.
Abstract: Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-A resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.
TL;DR: Interestingly, the genetically created hybrid protein (with a linear RGD sequence) showed similar or slightly better cell adhesion properties as the silk protein chemically modified with the cyclic RGD peptide.
TL;DR: A supramolecular approach to form the peptide amphiphile by connecting two building blocks through a non-covalent interaction would represent a major advance, especially in designing stimuliresponsive systems capable of being targeted by specific triggers.
Abstract: Single-tail peptide amphiphiles, have been explored as a new class of biomaterials in many fields including nanotechnology and tissue engineering. A typical peptide amphiphile molecule is linked through a covalent amide bond between a hydrophilic peptide sequence and a hydrophobic lipid of variable length. In an aqueous environment, these peptide amphiphiles undergo self-assembly into structures such as vesicles, both spherical and cylindrical micelles or nanotubes, and have been successfully applied in the biomedical sciences for biomaterial conjugation and as bioactive scaffolds for tissue engineering. Although covalent attachment of two components to form peptide amphiphiles has been extremely successful, the synthetic versatility and the ability to respond to external triggers remains limited. A supramolecular approach to form the peptide amphiphile by connecting two building blocks through a non-covalent interaction would represent a major advance, especially in designing stimuliresponsive systems capable of being targeted by specific triggers. Cucurbit[n]urils (CB[n]) are a family of macrocyclic hosts known to form inclusion complexes with selectivity and high binding affinity in aqueous media. One of the larger macrocycles in this family, CB[8], can be used as a “molecular handcuff” to join two molecules together in a non-covalent fashion, and has been applied to form biomaterials such as polymer–protein conjugation and protein dimerization. Additionally, CB[n] hosts have found great utility in “switch on/switch off” fluorescence assays by supramolecular complexation with various fluorescent guests. Pyrene and its derivatives have been widely used as fluorescence probes in a large number of complex systems, on account of their high fluorescence quantum yields, long excited state lifetimes and the ability to form excimers. Herein, we utilize a functional pyrene bearing an imidazolium group both as a fluorescence sensor and as a guest for CB[8] and linked it to a simple peptide sequence (1). Pyrene-functionalized peptide 1 is able to form the supramolecular peptide amphiphile complex 3 with viologen lipid 2 through CB[8] conjugation, as shown in Figure 1a. During the