scispace - formally typeset
Search or ask a question

Showing papers on "Peptide sequence published in 2015"


Journal ArticleDOI
09 Jan 2015-Science
TL;DR: SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, is identified as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases and Ragulator in an amino acid–sensitive fashion and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.
Abstract: The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

664 citations


Journal ArticleDOI
TL;DR: It is concluded that at least one-third of unassigned spectra arise from peptides with substoichiometric modifications, and an ultra-tolerant Sequest database search that allows peptide matching even with modifications of unknown masses is used.
Abstract: Fewer than half of all tandem mass spectrometry (MS/MS) spectra acquired in shotgun proteomics experiments are typically matched to a peptide with high confidence. Here we determine the identity of unassigned peptides using an ultra-tolerant Sequest database search that allows peptide matching even with modifications of unknown masses up to ± 500 Da. In a proteome-wide data set on HEK293 cells (9,513 proteins and 396,736 peptides), this approach matched an additional 184,000 modified peptides, which were linked to biological and chemical modifications representing 523 distinct mass bins, including phosphorylation, glycosylation and methylation. We localized all unknown modification masses to specific regions within a peptide. Known modifications were assigned to the correct amino acids with frequencies >90%. We conclude that at least one-third of unassigned spectra arise from peptides with substoichiometric modifications.

358 citations


Journal ArticleDOI
TL;DR: It is shown that intrinsically disordered, repeat proteins can be designed to exhibit tunable lower or upper critical solution temperature transitions in physiological solutions and that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition.
Abstract: Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level.

316 citations


Journal ArticleDOI
TL;DR: An updated version of the state-of-the-art NetMHCIIpan method with improved peptide binding register identification is described, which significantly outperformed the earlier 3.0 version.
Abstract: A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4+ T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1.

269 citations


Journal ArticleDOI
TL;DR: The model reveals that the individual layers of the Aβ fibril are formed by peptide dimers with face-to-face packing, explaining why aggregation inhibitors are most potent when targeting the C terminus, and explains how addition of C-terminal amino acids may stabilize peptide interaction.
Abstract: Alzheimer's disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of β-amyloid (Aβ) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aβ fibrillation is extensively documented, much is still unknown about the formation of these Aβ aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aβ(1-42), a particularly pathogenic variant of Aβ peptide. Our model reveals that the individual layers of the Aβ fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal β-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aβ peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aβ peptide.

195 citations


Journal ArticleDOI
01 Oct 2015-Gene
TL;DR: This article will specifically focus on the expression, function and regulation of Hsp90α, the stress inducible isoform of the molecular chaperone Hsp 90.

163 citations


Journal ArticleDOI
07 May 2015-Nature
TL;DR: It is shown that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.
Abstract: Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

146 citations


Journal ArticleDOI
TL;DR: The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity; it appears to be a conserved strategy in other peptide modifying enzymes.
Abstract: Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways, and many of the resulting products have potential as therapeutics. The ATP-dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have a conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate, suggesting that recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal that the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whereas catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity, and it appears to be a conserved strategy in other peptide-modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.

140 citations


Journal ArticleDOI
Jiao Tang1, Zhifu Han1, Yadong Sun1, Heqiao Zhang1, Xinqi Gong1, Jijie Chai1 
TL;DR: A crystal structure of the extracellular LRR domain of PEPR1 (PEPR1LRR) in complex with AtPep1 is reported, providing significant insight into prediction of recognition of other peptides by their cognate LRR-RKs.
Abstract: The endogenous peptides AtPep1-8 in Arabidopsis mature from the conserved C-terminal portions of their precursor proteins PROPEP1-8, respectively The two homologous leucine-rich repeat-receptor kinases (LRR-RKs) PEPR1 and PEPR2 act as receptors of AtPeps AtPep binding leads to stable association of PEPR1,2 with the shared receptor LRR-RK BAK1, eliciting immune responses similar to those induced by pathogens Here we report a crystal structure of the extracellular LRR domain of PEPR1 (PEPR1LRR) in complex with AtPep1 The structure reveals that AtPep1 adopts a fully extended conformation and binds to the inner surface of the superhelical PEPR1LRR Biochemical assays showed that AtPep1 is capable of inducing PEPR1LRR-BAK1LRR heterodimerization The conserved C-terminal portion of AtPep1 dominates AtPep1 binding to PEPR1LRR, with the last amino acid of AtPep1 Asn23 forming extensive interactions with PEPR1LRR Deletion of the last residue of AtPep1 significantly compromised AtPep1 interaction with PEPR1LRR Together, our data reveal a conserved structural mechanism of AtPep1 recognition by PEPR1, providing significant insight into prediction of recognition of other peptides by their cognate LRR-RKs

132 citations


Journal ArticleDOI
TL;DR: A highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues, achieving cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization.
Abstract: Macrocyclization is a valuable tool for drug design and protein engineering. Although various methods have been developed to prepare macrocycles, a general and efficient strategy is needed. Here we report a highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues. We achieved cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization. The reactions completed within minutes with up to 95% yields.

130 citations


Journal ArticleDOI
TL;DR: Analysis of the minimal inhibitory concentration revealed that DLP4 have antibacterial effects against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), and the amino acid sequence of the mature peptide was determined to be ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK.
Abstract: In this study, we induced and purified a novel antimicrobial peptide exhibiting activity against Gram-positive bacteria from the immunized hemolymph of Hermetia illucens larvae . The immunized hemolymph was extracted, and the novel defensin-like peptide 4 (DLP4) was purified using solid-phase extraction and reverse-phase chromatography. The purified DLP4 demonstrated a molecular weight of 4267 Da, as determined using the matrix-assisted laser desorption/ionization–time-of-flight (MALDI–TOF) method. From analysis of DLP4 by N-terminal amino acid sequencing using Edman degradation, combined with MALDI–TOF and rapid amplification of cDNA ends–polymerase chain reaction (RACE–PCR), the amino acid sequence of the mature peptide was determined to be ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK. In NCBI BLAST, the amino acid sequence of DPL4 was found to be 75% identical to the Phlebotomus duboscqi defensin. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP4 have antibacterial effects against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The expression of DLP4 transcripts in several tissues after bacterial challenge was measured by quantitative real-time PCR. Expression of the DLP4 gene hardly occurred throughout the body before immunization, but was mostly evident in the fat body after immunization.

Journal ArticleDOI
TL;DR: Analysis of thermodynamics data reported in the literature indicated that disease‐causing variants tend to destabilize proteins and their interactions, which prompted us to investigate the effects of amino acid mutations on large databases of experimentally measured energy changes in unrelated proteins.
Abstract: Statistical analysis was carried out on large set of naturally occurring human amino acid variations, and it was demonstrated that there is a preference for some amino acid substitutions to be associated with diseases. At an amino acid sequence level, it was shown that the disease-causing variants frequently involve drastic changes in amino acid physicochemical properties of proteins such as charge, hydrophobicity, and geometry. Structural analysis of variants involved in diseases and being frequently observed in human population showed similar trends: disease-causing variants tend to cause more changes in hydrogen bond network and salt bridges as compared with harmless amino acid mutations. Analysis of thermodynamics data reported in the literature, both experimental and computational, indicated that disease-causing variants tend to destabilize proteins and their interactions, which prompted us to investigate the effects of amino acid mutations on large databases of experimentally measured energy changes in unrelated proteins. Although the experimental datasets were linked neither to diseases nor exclusory to human proteins, the observed trends were the same: amino acid mutations tend to destabilize proteins and their interactions. Having in mind that structural and thermodynamics properties are interrelated, it is pointed out that any large change in any of them is anticipated to cause a disease.

Journal ArticleDOI
TL;DR: It is demonstrated how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide.
Abstract: Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation

Journal ArticleDOI
TL;DR: A genetically encodable, endosome escape-enhancing peptide that can substantially increase the cytoplasmic delivery of cationic proteins in vitro and in vivo is described.
Abstract: The inefficient delivery of proteins into mammalian cells remains a major barrier to realizing the therapeutic potential of many proteins. We and others have previously shown that superpositively charged proteins are efficiently endocytosed and can bring associated proteins and nucleic acids into cells. The vast majority of cargo delivered in this manner, however, remains in endosomes and does not reach the cytosol. In this study we designed and implemented a screen to discover peptides that enhance the endosomal escape of proteins fused to superpositively charged GFP (+36 GFP). From a screen of peptides previously reported to disrupt microbial membranes without known mammalian cell toxicity, we discovered a 13-residue peptide, aurein 1.2, that substantially increases cytosolic protein delivery by up to ∼5-fold in a cytosolic fractionation assay in cultured cells. Four additional independent assays for nonendosomal protein delivery collectively suggest that aurein 1.2 enhances endosomal escape of associated endocytosed protein cargo. Structure-function studies clarified peptide sequence and protein conjugation requirements for endosomal escape activity. When applied to the in vivo delivery of +36 GFP-Cre recombinase fusions into the inner ear of live mice, fusion with aurein 1.2 dramatically increased nonendosomal Cre recombinase delivery potency, resulting in up to 100% recombined inner hair cells and 96% recombined outer hair cells, compared to 0-4% recombined hair cells from +36-GFP-Cre without aurein 1.2. Collectively, these findings describe a genetically encodable, endosome escape-enhancing peptide that can substantially increase the cytoplasmic delivery of cationic proteins in vitro and in vivo.

Journal ArticleDOI
TL;DR: It is proposed that the proteasome's sequence preferences provide a second component to the degradation code and may fine-tune protein half-life in cells.
Abstract: The proteasome controls the concentrations of most proteins in eukaryotic cells. It recognizes its protein substrates through ubiquitin tags and initiates degradation at disordered regions within the substrate. Here we show that the proteasome has pronounced preferences for the amino acid sequence of the regions at which it initiates degradation. Specifically, proteins in which the initiation regions have biased amino acid compositions show longer half-lives in yeast than proteins with unbiased sequences in the regions. The relationship is also observed on a genomic scale in mouse cells. These preferences affect the degradation rates of proteins in vitro, can explain the unexpected stability of natural proteins in yeast and may affect the accumulation of toxic proteins in disease. We propose that the proteasome's sequence preferences provide a second component to the degradation code and may fine-tune protein half-life in cells.

Journal ArticleDOI
TL;DR: Reaction network starting from monomer mixtures of Aβ40 and Aβ42 accelerates A β40 fibril formation and separate fibrils form as secondary nucleation and elongation are highly specific.
Abstract: The assembly of proteins into amyloid fibrils, a phenomenon central to several currently incurable human diseases, is a process of high specificity that commonly tolerates only a low level of sequence mismatch in the component polypeptides. However, in many cases aggregation-prone polypeptides exist as mixtures with variations in sequence length or post-translational modifications; in particular amyloid β (Aβ) peptides of variable length coexist in the central nervous system and possess a propensity to aggregate in Alzheimer's disease and related dementias. Here we have probed the co-aggregation and cross-seeding behavior of the two principal forms of Aβ, Aβ40 and Aβ42 that differ by two hydrophobic residues at the C-terminus. We find, using isotope-labeling, mass spectrometry and electron microscopy that they separate preferentially into homomolecular pure Aβ42 and Aβ40 structures during fibril formation from mixed solutions of both peptides. Although mixed fibrils are not formed, the kinetics of amyloid formation of one peptide is affected by the presence of the other form. In particular monomeric Aβ42 accelerates strongly the aggregation of Aβ40 in a concentration-dependent manner. Whereas the aggregation of each peptide is catalyzed by low concentrations of preformed fibrils of the same peptide, we observe a comparably insignificant effect when Aβ42 fibrils are added to Aβ40 monomer or vice versa. Therefore we conclude that fibril-catalysed nucleus formation and elongation are highly sequence specific events but Aβ40 and Aβ42 interact during primary nucleation. These results provide a molecular level description of homomolecular and heteromolecular aggregation steps in mixtures of polypeptide sequence variants.

Journal ArticleDOI
TL;DR: A combined chemical and biological route to the complex peptide substrate, relying on chemical synthesis of a modified C-terminal fragment and coupling to a 38-residue leader peptide by means of native chemical ligation (NCL), provides a new chemoenzymatic route to this promising class of antibiotics.
Abstract: Thiocillins from Bacillus cereus ATCC 14579 are members of the well-known thiazolyl peptide class of natural product antibiotics, the biosynthesis of which has recently been shown to proceed via post-translational modification of ribosomally encoded precursor peptides. It has long been hypothesized that the final step of thiazolyl peptide biosynthesis involves a formal [4 + 2] cycloaddition between two dehydroalanines, a unique transformation that had eluded enzymatic characterization. Here we demonstrate that TclM, a single enzyme from the thiocillin biosynthetic pathway, catalyzes this transformation. To facilitate characterization of this new class of enzyme, we have developed a combined chemical and biological route to the complex peptide substrate, relying on chemical synthesis of a modified C-terminal fragment and coupling to a 38-residue leader peptide by means of native chemical ligation (NCL). This strategy, combined with active enzyme, provides a new chemoenzymatic route to this promising class ...

Journal ArticleDOI
TL;DR: The data show that FYCO1 requires a functional LIR motif to facilitate efficient maturation of autophagosomes under basal conditions, whereas starvation-induced autophagy was unaffected.

Journal ArticleDOI
TL;DR: Human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5'-phosphate (R5P) tags covalently attached to the modified proteins.
Abstract: ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro , converting it into ribose-5′-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS.

Journal ArticleDOI
TL;DR: The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.

Journal ArticleDOI
TL;DR: It is found that certain molecules containing a "penta-arg" motif reach the cytosol, intact, with efficiencies greater than 50%, which is at least 10-fold higher than that observed for the widely studied cationic sequence derived from HIV Tat or polyarginine Arg8.
Abstract: We used fluorescence correlation spectroscopy (FCS) to accurately and precisely determine the relative efficiencies with which three families of "cell-penetrating peptides" traffic to the cytosol of mammalian cells. We find that certain molecules containing a "penta-arg" motif reach the cytosol, intact, with efficiencies greater than 50%. This value is at least 10-fold higher than that observed for the widely studied cationic sequence derived from HIV Tat or polyarginine Arg8, and equals that of hydrocarbon-stapled peptides that are active in cells and animals. Moreover, we show that the efficiency with which stapled peptides reach the cytosol, as determined by FCS, correlates directly with their efficacy in cell-based assays. We expect that these findings and the associated technology will aid the design of peptides, proteins, and peptide mimetics that predictably and efficiently reach the interior of mammalian cells.

Journal ArticleDOI
TL;DR: Both in vitro and in vivo studies confirm the chimeric peptide‐based nanocarrier with a good synergistic effect is a promising nanoplatform for tumor treatments.
Abstract: In this study, a pH sensitive chimeric peptide is developed to codeliver a photosensitizer, protoporphyrin IX (PpIX), and plasmid DNA simultaneously. In the presence of matrix metalloproteinase-2 (MMP-2), the chimeric peptide undergoes the process of hydrolysis of PLGVR peptide sequence, exfoliation of PEG, and increase of positive charges. As a result, the chimeric peptide can be preferentially uptaken by MMP-2 rich tumor cells. To realize synergistic effect of drug and gene delivery, a dual-stage light irradiation strategy is developed, i.e., the short time light irradiation can efficiently enhance the endosomal escape of the chimeric peptide/PpIX/DNA complexes by the formation the reactive oxygen species (ROS), resulting in synergistic endosomal escape and improved DNA expression. In addition, due to the screened phototoxicity of PpIX, short time light irradiation does not lead to detectable changes in the cell viability. After the gene transfection, the screened phototoxicity of PpIX is subsequently stimulated by long time irradiation to achieve high synergistic efficacy of photodynamic and gene therapies. Both in vitro and in vivo studies confirm the chimeric peptide-based nanocarrier with a good synergistic effect is a promising nanoplatform for tumor treatments.

Journal ArticleDOI
TL;DR: It is reported that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice and may provide a significant additional benefit when this peptide is used for drug delivery to tumors.
Abstract: Tumor-specific tissue-penetrating peptides deliver drugs into extravascular tumor tissue by increasing tumor vascular permeability through interaction with neuropilin (NRP). Here we report that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice. The anti-metastatic effect was mediated by the NRP-binding RXXK peptide motif (CendR motif), and not by the integrin-binding RGD motif. iRGD inhibited migration of tumor cells and caused chemorepulsion in vitro in a CendR- and NRP-1-dependent manner. The peptide induced dramatic collapse of cellular processes and partial cell detachment, resulting in the repellent activity. These effects were prominently displayed when the cells were seeded on fibronectin, suggesting a role of CendR in functional regulation of integrins. The anti-metastatic activity of iRGD may provide a significant additional benefit when this peptide is used for drug delivery to tumors.

Journal ArticleDOI
TL;DR: It is found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant, Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.
Abstract: The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein’s assembly, implying that BamD’s interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.

Journal ArticleDOI
TL;DR: Key roles for residues conserved between bacterial and eukaryotic homologues suggest a conserved mechanism of peptide recognition and transport that in some cases has been subtly modified in individual species.

Journal ArticleDOI
Ye Huijun1, Libing Wang1, Renliang Huang1, Rongxin Su1, Boshi Liu1, Wei Qi1, Zhimin He1 
TL;DR: Findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.
Abstract: The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

Journal ArticleDOI
TL;DR: A strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold that should enable the design of biostable α/ β- peptides that bind tightly and specifically to diverse targets of biomedical interest.
Abstract: Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165 -induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

Journal ArticleDOI
TL;DR: A series of imperfect amphipathic peptides designed by placing different types of amino acid residues at the hydrogen bond linked positions of α-helix structures to characterize their antimicrobial properties and mechanism of action exhibited excellent antimicrobial potency.

Journal ArticleDOI
TL;DR: It was found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect.
Abstract: The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α-amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect.

Journal ArticleDOI
TL;DR: The SMART model has been introduced to cover the full range of possibilities of novel antimicrobial compounds designed from amphipathic polymers, peptide mimetics, combinations of ultra‐short polypeptides with hydrophobic anchors or small designer molecules.
Abstract: Biophysical and structural studies of peptide–lipid interactions, peptide topology and dynamics have changed our view on how antimicrobial peptides insert and interact with membranes. Clearly, both the peptides and the lipids are highly dynamic, change and mutually adapt their conformation, membrane penetration and detailed morphology on a local and a global level. As a consequence, the peptides and lipids can form a wide variety of supramolecular assemblies in which the more hydrophobic sequences preferentially, but not exclusively, adopt transmembrane alignments and have the potential to form oligomeric structures similar to those suggested by the transmembrane helical bundle model. In contrast, charged amphipathic sequences tend to stay intercalated at the membrane interface where they cause pronounced disruptions of the phospholipid fatty acyl packing. At increasing local or global concentrations, the peptides result in transient membrane openings, rupture and ultimately lysis. Depending on peptide-to-lipid ratio, lipid composition and environmental factors (temperature, buffer composition, ionic strength, etc.), the same peptide sequence can result in a variety of those responses. Therefore, the SMART model has been introduced to cover the full range of possibilities. With such a view in mind, novel antimicrobial compounds have been designed from amphipathic polymers, peptide mimetics, combinations of ultra-short polypeptides with hydrophobic anchors or small designer molecules. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.