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At least 145 records · Page 8

All sources NASA NTRS DOE OSTI

DOE OSTI2023

Developing a peptide concatemer (PepCon) as a process control for LC-MS based proteomics

Quantitative concatenated peptide (QconCAT) technology is currently used for the absolute quantification of proteins of interest in a biological sample. It relies on artificially created proteins that are concatenations of different, isotopically labeled peptides. Because these isotopically labeled peptides are at a 1:1 ratio and correspond to naturally occurring peptides in the biological sample, each peptide can be used as a standard for the absolute quantitation of all proteins of interest at once. Although the QconCAT technology has utility for quantitation of known peptides in a mixture, it is not helpful for scientists who need a proteomics standard (1) that can be spiked into a protein mixture at an extremely low level, (2) that can be co-purified during sample fractionation, and (3) that is optimized for ESI used in mass spectrometry. Thus, there exists a need for an ideal standard protein that is large enough to behave as a protein but consists of multiple, concatenated copies of the same peptide, which, upon digestion, amplifies (e.g., >10-fold) into a detectable peptide species. Here, this technical report describes the development of “PepCon”, a peptide concatemer that fulfills this unmet need.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Simplification of the purification of heat stable recombinant low molecular weight proteins and peptides from GST-fusion products

The synthesis and purification of peptides of importance in the fields of research and medicine continue to be a challenging task. Chemical synthesis of oligopeptides, especially those greater than 25 amino acids, is cost prohibitive. On the other hand, several bottlenecks exist in the production of recombinant short peptides in heterologous expression hosts such as Escherichia coli (E. coli). In this paper, a rapid, cost-effective, and reliable method for the production and singlestep-purification of peptides and small proteins was developed. Five peptides and small proteins were overexpressed in E. coli as GST-fusion products in high yields. The recombinant peptides or proteins were successfully purified after enzymatic cleavage with selective heat-induced precipitation of the GST-affinity tag. Qualitative and quantitative analysis using SDS-PAGE and mass spectrometric methods suggest that the recombinant peptides/ proteins were purified to >95% homogeneity. Results of biophysical experiments, including multi-dimensional NMR spectroscopy, show that the purified proteins/ peptides retain their native conformation. Isothermal titration calorimetry studies indicate no significant change in the binding affinity of the heattreated purified product to their interacting partner(s) compared to the recombinant peptides purified by conventional chromatographic procedures without subjecting to heat treatment. In our opinion, the results reported render the purification of recombinant proteins/ peptides of biomedical relevance using our proposed method easy and reliable.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2013

Solvation Free Energies of Alanine Peptides: The Effect of Flexibility

The electrostatic (?Gel), cavity-formation (?Gvdw), and total (?G) solvation free energies for 10 alanine peptides ranging in length (n) from 1 to 10 monomers were calculated. The free energies were computed both with xed, extended conformations of the peptides and again for some of the peptides without constraints. The solvation free energies, ?Gel, ?Gvdw, and ?G, were found to be linear in n, with the slopes of the best-fit lines being gamma_el, gamma_vdw, and gamma, respectively. Both gamma_el and gamma were negative for fixed and flexible peptides, and gamma_vdw was negative for fixed peptides. That gamma_vdw was negative was surprising, as experimental data on alkanes, theoretical models, and MD computations on small molecules and model systems generally suggest that gamma_vdw should be positive. A negative gamma_vdw seemingly contradicts the notion that ?Gvdw drives the initial collapse of the protein when it folds by favoring conformations with small surface areas, but when we computed ?Gvdw for the flexible peptides, thereby allowing the peptides to assume natural ensembles of more compact conformations, gamma-vdw was positive. Because most proteins do not assume extended conformations, a ?Gvdw that increases with increasing surface area may be typical for globular proteins. An alternative hypothesis is that the collapse is driven by intramolecular interactions. We show that the intramolecular van der Waal's interaction energy is more favorable for the flexible than for the extended peptides, seemingly favoring this hypothesis, but the large fluctuations in this energy may make attributing the collapse of the peptide to this intramolecular energy difficult.

Environmental Molecular Sciences Laboratory↗

DOE OSTI2021

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

De novo design of D-peptide ligands: Application to influenza virus hemagglutinin

D-peptides hold great promise as therapeutics by alleviating the challenges of metabolic stability and immunogenicity in L-peptides. However, current D-peptide discovery methods are severely limited by specific size, structure, and the chemical synthesizability of their protein targets. Here, we describe a computational method for de novo design of D-peptides that bind to an epitope of interest on the target protein using Rosetta’s hotspot-centric approach. The approach comprises identifying hotspot sidechains in a functional protein–protein interaction and grafting these side chains onto much smaller structured peptide scaffolds of opposite chirality. The approach enables more facile design of D-peptides and its applicability is demonstrated by design of D-peptidic binders of influenza A virus hemagglutinin, resulting in identification of multiple D-peptide lead series. The X-ray structure of one of the leads at 2.38 Å resolution verifies the validity of the approach. This method should be generally applicable to targets with detailed structural information, independent of molecular size, and accelerate development of stable, peptide-based therapeutics.

Science & Technology - Other Topics↗

DOE OSTI2019

ERAP1 enzyme-mediated trimming and structural analyses of MHC I–bound precursor peptides yield novel insights into antigen processing and presentation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 critically shape the major histocompatibility complex I (MHC I) immunopeptidome. The ERAPs remove N-terminal residues from antigenic precursor peptides and generate optimal-length peptides (i.e. 8–10-mers) to fit into the MHC class I groove. It is therefore intriguing that MHC class I molecules can present N-terminally extended peptides on the cell surface that can elicit CD8+ T-cell responses. This observation likely reflects gaps in our understanding of how antigens are processed by the ERAP enzymes. To better understand ERAPs' function in antigen processing, here we generated a nested set of N-terminally extended 10–20-mer peptides (RA)nAAKKKYCL covalently bound to the human leukocyte antigen (HLA)-B*0801. We used X-ray crystallography, thermostability assessments, and an ERAP1-trimming assay to characterize these complexes. The X-ray structures determined at 1.40–1.65 Å resolutions revealed that the residue extensions (RA)n unexpectedly protrude out of the A pocket of HLA-B*0801, whereas the AAKKKYCL core of all peptides adopts similar, bound conformations. HLA-B*0801 residue 62 was critical to open the A pocket. We also show that HLA-B*0801 and antigenic precursor peptides form stable complexes. Finally, ERAP1-mediated trimming of the MHC I–bound peptides required a minimal length of 14 amino acids. We propose a mechanistic model explaining how ERAP1-mediated trimming of MHC I–bound peptides in cells can generate peptides of canonical as well as noncanonical lengths that still serve as stable MHC I ligands. Our results provide a framework to better understand how the ERAP enzymes influence the MHC I immunopeptidome.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

A short synthetic peptide fragment of human C2ORF40 has therapeutic potential in breast cancer

C2ORF40 encodes a secreted protein which is cleaved to generate soluble peptides by proteolytic processing and this process is believed to be necessary for C2ORF40 to exert cell type specific biological activity. Here, we reported a short mimic peptide of human C2ORF40 acts potential therapeutic efficacy in human cancer cells in vitro and in vivo. We synthesized a short peptide of human C2ORF40, named C2ORF40 mimic peptide fragment and assessed its biological function on cancer cell growth, migration and tumorigenesis. Cell growth assay showed that C2ORF40 mimic peptide fragment significantly suppressed cell proliferation of breast and lung cancer cells. Moreover, C2ORF40 mimic peptide fragment significantly inhibited the migration and invasion of breast cancer cells. Furthermore, we showed that this peptide suppressed tumorigenesis in breast tumor xenograft model. Cell cycle assay indicated that the C2ORF40 mimic peptide fragment suppressed the growth of tumor cells through inducing mitotic phase arrest. In conclusion, our results firstly suggested that this short synthetic peptide of human C2ORF40 may be a candidate tumor therapeutic agent.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2019

Improved Modeling of Peptide-Protein Binding Through Global Docking and Accelerated Molecular Dynamics Simulations

We find that peptides mediate up to 40% of known protein-protein interactions in higher eukaryotes and play a key role in cellular signaling, protein trafficking, immunology, and oncology. However, it is challenging to predict peptide-protein binding with conventional computational modeling approaches, due to slow dynamics and high peptide flexibility. Here, we present a prototype of the approach which combines global peptide docking using ClusPro PeptiDock and all-atom enhanced simulations using Gaussian accelerated molecular dynamics (GaMD). For three distinct model peptides, the lowest backbone root-mean-square deviations (RMSDs) of their bound conformations relative to X-ray structures obtained from PeptiDock were 3.3–4.8 Å, being medium quality predictions according to the Critical Assessment of PRediction of Interactions (CAPRI) criteria. GaMD simulations refined the peptide-protein complex structures with significantly reduced peptide backbone RMSDs of 0.6–2.7 Å, yielding two high quality (sub-angstrom) and one medium quality models. Furthermore, the GaMD simulations identified important low-energy conformational states and revealed the mechanism of peptide binding to the target proteins. Therefore, PeptiDock+GaMD is a promising approach for exploring peptide-protein interactions.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1996

Transmembrane transport of peptide type compounds: prospects for oral delivery

Synthesis and delivery of potential therapeutic peptides and peptidomimetic compounds has been the focus of intense research over the last 10 years. While it is widely recognized that numerous limitations apply to oral delivery of peptides, some of the limiting factors have been addressed and their mechanisms elucidated, which has lead to promising strategies. This article will briefly summarize the challenges, results and current approaches of oral peptide delivery and give some insight on future strategies. The barriers determining peptide bioavailability after oral administration are intestinal membrane permability, size limitations, intestinal and hepatic metabolism and in some cases solubility limitations. Poor membrane permeabilities of hydrophilic peptides might be overcome by structurally modifying the compounds, thus increasing their membrane partition characteristics and/or their affinity to carrier proteins. Another approach is the site-specific delivery of the peptide to the most permeable parts of the intestine. The current view on size limitation for oral drug delivery has neglected partition considerations. Recent studies suggest that compounds with a molecular weight up to 4000 might be significantly absorbed, assuming appropriate partition behavior and stability. Metabolism, probably the most significant factor in the absorption fate of peptides, might be controlled by coadministration of competitive enzyme inhibitors, structural modifications and administration of the compound as a well absorbed prodrug that is converted into the therapeutically active agent after its absorption. For some peptides poor solubility might present a limitation to oral absorption, an issue that has been addressed by mechanistically defining and therefore improving formulation parameters. Effective oral peptide delivery requires further development in understanding these complex mechanisms in order to maximize the therapeutic potential of this class of compounds.

NASA Discipline Regulatory Physiology↗

DOE OSTI2016

Use of Galerina marginata genes and proteins for peptide production

The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2017

Use of Galerina marginata genes and proteins for peptide production

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Use of galerina marginata genes and proteins for peptide production

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Self-assembling peptide-based building blocks in medical applications

Peptides and peptide-conjugates, comprising natural and synthetic building blocks, are an increasingly popular class of biomaterials. Self-assembled nanostructures based on peptides and peptide-conjugates offer advantages such as precise selectivity and multifunctionality that can address challenges and limitations in the clinic. In this review article, we discuss recent developments in the design and self-assembly of various nanomaterials based on peptides and peptide-conjugates for medical applications, and categorize them into two themes based on the driving forces of molecular self-assembly. First, we present the self-assembled nanostructures driven by the supramolecular interactions between the peptides, with or without the presence of conjugates. The studies where nanoassembly is driven by the interactions between the conjugates of peptide-conjugates are then presented. Particular emphasis is given to in vivo studies focusing on therapeutics, diagnostics, immune modulation and regenerative medicine. Finally, challenges and future perspectives are presented.

Medicine↗

DOE OSTI2022

Mechanism of Peptide Agonist Binding in CXCR4 Chemokine Receptor

Chemokine receptors are key G-protein-coupled receptors (GPCRs) that control cell migration in immune system responses, development of cardiovascular and central nervous systems, and numerous diseases. In particular, the CXCR4 chemokine receptor promotes metastasis, tumor growth and angiogenesis in cancers. CXCR4 is also used as one of the two co-receptors for T-tropic HIV-1 entry into host cells. Therefore, CXCR4 serves as an important therapeutic target for treating cancers and HIV infection. Apart from the CXCL12 endogenous peptide agonist, previous studies suggested that the first 17 amino acids of CXCL12 are sufficient to activate CXCR4. Two 17-residue peptides with positions 1–4 mutated to RSVM and ASLW functioned as super and partial agonists of CXCR4, respectively. However, the mechanism of peptide agonist binding in CXCR4 remains unclear. Here, we have investigated this mechanism through all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method. The Pep-GaMD simulations have allowed us to explore representative binding conformations of each peptide and identify critical low-energy states of CXCR4 activated by the super versus partial peptide agonists. Our simulations have provided important mechanistic insights into peptide agonist binding in CXCR4, which are expected to facilitate rational design of new peptide modulators of CXCR4 and other chemokine receptors.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

An exhaustive survey of regular peptide conformations using a new metric for backbone handedness ( h )

The Ramachandran plot is important to structural biology as it describes a peptide backbone in the context of its dominant degrees of freedom—the backbone dihedral angles φ and ψ (Ramachandran, Ramakrishnan & Sasisekharan, 1963). Since its introduction, the Ramachandran plot has been a crucial tool to characterize protein backbone features. However, the conformation or twist of a backbone as a function of φ and ψ has not been completely described for both cis and trans backbones. Additionally, little intuitive understanding is available about a peptide’s conformation simply from knowing the φ and ψ values of a peptide (e.g., is the regular peptide defined by φ = ψ = - 100° left-handed or right-handed?). This report provides a new metric for backbone handedness ( h ) based on interpreting a peptide backbone as a helix with axial displacement d and angular displacement θ , both of which are derived from a peptide backbone’s internal coordinates, especially dihedral angles φ , ψ and ω . In particular, h equals sin( θ ) d / d |, with range [-1, 1] and negative (or positive) values indicating left(or right)-handedness. The metric h is used to characterize the handedness of every region of the Ramachandran plot for both cis ( ω = 0°) and trans ( ω = 180°) backbones, which provides the first exhaustive survey of twist handedness in Ramachandran ( φ , ψ ) space. These maps fill in the ‘dead space’ within the Ramachandran plot, which are regions that are not commonly accessed by structured proteins, but which may be accessible to intrinsically disordered proteins, short peptide fragments, and protein mimics such as peptoids. Finally, building on the work of (Zacharias & Knapp, 2013), this report presents a new plot based on d and θ that serves as a universal and intuitive alternative to the Ramachandran plot. The universality arises from the fact that the co-inhabitants of such a plot include every possible peptide backbone including cis and trans backbones. The intuitiveness arises from the fact that d and θ provide, at a glance, numerous aspects of the backbone including compactness, handedness, and planarity.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Signatures of Mechanically Interlocked Topology of Lasso Peptides by Ion Mobility–Mass Spectrometry: Lessons from a Collection of Representatives

Lasso peptides are characterized by a mechanically interlocked structure, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Their compact and stable structures have a significant impact on their biological and physical properties and make them highly interesting for drug development. Ion mobility - mass spectrometry (IM-MS) has shown to be effective to discriminate the lasso topology from their corresponding branched-cyclic topoisomers in which the C-terminal tail is unthreaded. In fact, previous comparison of the IM-MS data of the two topologies has yielded three trends that allow differentiation of the lasso fold from the branched-cyclic structure: (1) the low abundance of highly charged ions, (2) the low change in collision cross sections (CCS) with increasing charge state and (3) a narrow ion mobility peak width. In this study, a three-dimensional plot was generated using three indicators based on these three trends: (1) mean charge divided by mass (ζ), (2) relative range of CCS covered by all protonated molecules (ΔΩ/Ω) and (3) mean ion mobility peak width (δΩ). The data were first collected on a set of twenty one lasso peptides and eight branched-cyclic peptides. The indicators were obtained also for eight variants of the well-known lasso peptide MccJ25 obtained by site-directed mutagenesis and further extended to five linear peptides, two macrocyclic peptides and one disulfide constrained peptide. In all cases, a clear clustering was observed between constrained and unconstrained structures, thus providing a new strategy to discriminate mechanically interlocked topologies. .

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2016

Designer interface peptide grafts target estrogen receptor alpha dimerization

The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide “I-box” derived from ER residues 503–518 specifically blocks ER dimerization. Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479–485), LQQQHQRLAQ (residues 497–506), and LSHIRHMSNK (residues 511–520) and reported the suitability of using LQQQHQRLAQ (ER 497–506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. - Highlights: • Designer peptide grafts retain core molecular recognition motif during MD simulations. • Designer peptide grafts with Poly-ALA helix form stable complexes with estrogen receptor in silico. • Inhibitor peptides significantly decrease estrogen induced cell proliferation of ER positive breast cancer cells in vitro.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Chloroplast stromal processing peptidase activity is modulated by transit peptide determinants that include inhibitory roles for its N-terminal domain and initial Met

Highlights: • Inactivation of SPP is not through a “cysteine switch” mechanism. • Thiol-modifying reagents do not stimulate preSPP activity but diminish mature SPP activity. • NEM disrupts both substrate binding and peptide bond cleavage by SPP. • Deletion of N terminus of preSPP transit peptide including initial Met triggers processing activity. • The active preSPP deletion mutants no longer possess predicted Hsp70 binding sites. The stromal processing peptidase (SPP) removes transit peptides as precursor proteins enter the chloroplast and different plastid types. SPP is synthesized as a latent, inactive precursor (preSPP) with an atypically long transit peptide. Determinants in the pea (Pisum sativum) SPP transit peptide that regulate mature SPP activation were investigated. Mutational and chemical analyses with protein modifying agents (NEM and APMA) showed a conserved transit peptide Cys-X-Ser/Thr-Cys motif did not inhibit SPP via a “cysteine switch” mechanism through His-X-X-Glu-His site interactions, although cysteines in mature SPP contribute to an active conformation. Significantly, a transit peptide deletion of only the N-terminal 28 amino acids activates SPP located downstream. Short deletions within this region suggest removal of the initial Met plays a pivotal, mechanistic role. Other deletions of ∼30 amino acids along the length of the transit peptide do not individually trigger activity, but larger deletions including Met have an additive effect indicating its removal may be a critical early step during preSPP import. Interestingly, the active preSPP deletion mutants no longer possess predicted Hsp70 binding sites including initial Met, thus Hsp70 interactions may restrict SPP from attaining an active conformation.

60 APPLIED LIFE SCIENCES↗