Hans Ippel

Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5

Disrupting the HNP1 and CCL5 heteromer between neutrophils and platelets blocks monocyte recruitment to inflammatory sites. Anti-inflammatory reaches for the SKY Inflammation aids the body’s response to infection or injury, but can cause damage if excessive or unresolved. Alard et al. now examine how two early inflammatory mediators—neutrophils and platelets—cooperate to enhance inflammation. They found that human neutrophil peptide 1 (HNP1), which is secreted from neutrophils, forms a heteromer with CCL5 on platelets, resulting in stimulated monocyte adhesion and an increase in inflammation. Disrupting this interaction with a peptide (SKY) decreased inflammation and blocked monocyte recruitment in a mouse model of myocardial infarction. If these results hold true in humans, they could form the basis for a new specific therapeutic in inflammation-associated diseases. In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation

Functional synergism and inhibitory effects of chemokine heterodimers can be selectively targeted by specific peptides in models of inflammation. Hampering heterodimers interrupts inflammation Inflammation is dependent on the recruitment of cells responding to chemokines. Von Hundelshausen et al. cataloged how human chemokines interact with each other and found that certain kinds of chemokine pairs can activate or inhibit receptor signaling. These chemokine heterodimers were shown to be active in mouse models of acute and chronic inflammation, which were ameliorated by treatment with a peptide designed to disrupt the chemokine pairing. Patients suffering from inflammatory conditions such as atherosclerosis could benefit from these kinds of therapeutics. Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.

Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR

Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1-111) and the conserved carbohydrate recognition domain (CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete (13)C/(15)N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded β-sheet behind the canonical carbohydrate-binding 6-stranded β-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)…P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for β-galactosides.

Binding of polysaccharides to human galectin-3 at a noncanonical site in its carbohydrate recognition domain

Galectin-3 (Gal-3) is a multifunctional lectin, unique to galectins by the presence of a long N-terminal tail (NT) off of its carbohydrate recognition domain (CRD). Many previous studies have investigated binding of small carbohydrates to its CRD. Here, we used nuclear magnetic resonance spectroscopy ((15)N-(1)H heteronuclear single quantum coherence data) to assess binding of (15)N-Gal-3 (and truncated (15)N-Gal-3 CRD) to several, relatively large polysaccharides, including eight varieties of galactomannans (GMs), as well as a β(1 → 4)-polymannan and an α-branched mannan. Overall, we found that these polysaccharides with a larger carbohydrate footprint interact primarily with a noncanonical carbohydrate-binding site on the F-face of the Gal-3 CRD β-sandwich, and to a less extent, if at all, with the canonical carbohydrate-binding site on the S-face. While there is no evidence for interaction with the NT itself, it does appear that the NT somehow mediates stronger interactions between the Gal-3 CRD and the GMs. Significant Gal-3 resonance broadening observed during polysaccharide titrations indicates that interactions occur in the intermediate exchange regime, and analysis of these data allows estimation of affinities and stoichiometries that range from 4 × 10(4) to 12 × 10(4) M(-1) per site and multiple sites per polysaccharide, respectively. We also found that lactose can still bind to the CRD S-face of GM-bound Gal-3, with the binding of one ligand attenuating affinity of the other. These data are compared with previous results on Gal-1, revealing differences and similarities. They also provide research direction to the development of these polysaccharides as galectin-targeting therapeutics in the clinic.

A novel approach for the intravenous delivery of leuprolide using core-cross-linked polymeric micelles

Therapeutic peptides are highly attractive drugs for the treatment of various diseases. However, their poor pharmacokinetics due to rapid renal elimination limits their clinical applications. In this study, a model hormone peptide, leuprolide, was covalently linked to core-cross-linked polymeric micelles (CCL-PMs) via two different hydrolysable ester linkages, thereby yielding a nanoparticulate system with tuneable drug release kinetics. The ester linkage that provided the slowest peptide release kinetics was selected for in vivo evaluation. Compared to the soluble peptide, the leuprolide-entrapped CCL-PMs showed a prolonged circulation half-life (14.4h) following a single intravenous injection in healthy rats and the released leuprolide was detected in blood for 3days. In addition, the area under the plasma concentration-time curve (AUC) value was >100-fold higher for leuprolide-entrapped CCL-PMs than for soluble leuprolide. Importantly, the released peptide remained biologically active as demonstrated by increased and long-lasting plasma testosterone levels. This study shows that covalent linkage of peptides to CCL-PMs via hydrolytically sensitive ester bonds is a promising approach to achieving sustained systemic levels of peptides after intravenous administration.

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with (15)N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

Chemoselective Oxime Reactions in Proteins and Peptides by Using an Optimized Oxime Strategy: The Demise of Levulinic Acid

form a special imine known as an oxime (3). Advantages of this strategy include chemoselectivity, as a ketone is inert to most other reactions, and the mild conditions in which the reaction can be performed. Oxime formation has been thoroughly investigated and was found to proceed in a step-wise manner, depending on pH. Although the reaction proceeds faster in acidic conditions (pH 4–5), oximes will also form at neutral pH. Furthermore, oxime formation can be accelerated by addition of the catalyst aniline, and oxime linkages are stable at neutral pH. Taken together, these properties make the oxime linkage one of the preferred methods for the chemoselective modification of peptides and proteins. Levulinic acid (LA) is one of the most frequently used ketones for oxime formation, and is generally introduced by attachment to a lysine side chain e-NH2 or the N-terminal NH2 moiety. The reaction between the protein–LA complex and an aminooxy moiety proceeds well at millimolar concentrations; however, the relatively low quantity and high molecular weight of most proteins result in sub-millimolar protein concentrations and limitations of the oxime reaction. Under these conditions, we and others have found that oxime reaction yields are low because of the formation of a levulinoyl-derived side-product that competes with oxime bond formation. As oxime ligations are increasingly attractive and provide an orthogonal approach to label proteins, we explored alternative ketone moieties for bioconjugation. Our hypothesis was that the levulinoyl side-product (corresponding to a mass loss of 18 Da) is the result of intramolecular cyclization of LA, thus preventing the LA ketone group from reacting with the aminooxy moiety. The formation of this by-product is mainly seen at low concentrations, because under these conditions the concentration-independent cyclization side-reaction benefits from the slow oxime formation. To study the molecular mechanism underlying levulinoyl cyclization, the pentapeptide LYRAK was synthesized with LA coupled to the lysine e-amine (LYRAK(LA)). Lyophilized LYRAK(LA) was dissolved in water under acidic conditions (pH 4.5) and was left to cyclize spontaneously for 72 h at room temperature. The conversion to cyclized derivatives was monitored by ESI-MS and NMR (both in [D6]DMSO and D2O) and structurally characterized by 2D NMR experiments (Figures S1 and S2). The cyclization of LA was tested at three concentrations and was shown to be concentration independent (Figure S3). Based on these results, the following reaction mechanism for intramolecular cyclization of the levulinoyl moiety was proposed (Scheme 2). The amide nitrogen in the linear levulinoyl peptide 4 performs a nucleophilic attack on the ketone carbonyl group resulting in the cyclic intermediate 5. This reaction was previously reported to be favored by protonation of the carbonyl. Spontaneous dehydration of 5 leads to the iminium species 6, which is stabilized by TFA in the solution. Spontaneous isomerization of 6 leads to 7 and 8, as can determined by 2D NMR methods (Figure S1). Conversion from 5 to the exocyclic-double-bond-containing species 7 has been reported, but formation of the endocyclic double bond has not. Here, 8 was observed but it was formed as a minor product compared Scheme 1. Overall oxime reaction. A ketone or aldehyde (1) reacts with an aminooxy (2) to form an oxime bond (3).

Probing Functional Heteromeric Chemokine Protein-Protein Interactions through Conformation-Assisted Oxime Ligation

Abstract Protein–protein interactions (PPIs) govern most processes in living cells. Current drug development strategies are aimed at disrupting or stabilizing PPIs, which require a thorough understanding of PPI mechanisms. Examples of such PPIs are heteromeric chemokine interactions that are potentially involved in pathological disorders such as cancer, atherosclerosis, and HIV. It remains unclear whether this functional modulation is mediated by heterodimer formation or by the additive effects of mixed chemokines on their respective receptors. To address this issue, we report the synthesis of a covalent RANTES‐PF4 heterodimer (termed OPRAH) by total chemical synthesis and oxime ligation, with an acceleration of the final ligation step driven by PPIs between RANTES and PF4. Compared to mixed separate chemokines, OPRAH exhibited increased biological activity, thus providing evidence that physical formation of the heterodimer indeed mediates enhanced function.

Optical signature of nerve tissue-Exploratory ex vivo study comparing optical, histological, and molecular characteristics of different adipose and nerve tissues: OPTICAL SIGNATURE OF NERVE TISSUE

During several anesthesiological procedures, needles are inserted through the skin of a patient to target nerves. In most cases, the needle traverses several tissues—skin, subcutaneous adipose tissue, muscles, nerves, and blood vessels—to reach the target nerve. A clear identification of the target nerve can improve the success of the nerve block and reduce the rate of complications. This may be accomplished with diffuse reflectance spectroscopy (DRS) which can provide a quantitative measure of the tissue composition. The goal of the current study was to further explore the morphological, biological, chemical, and optical characteristics of the tissues encountered during needle insertion to improve future DRS classification algorithms.

A One-Pot “Triple-C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides

A broadly applicable one‐pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)‐catalyzed reaction (CEPS/CLIPS/CuAAC; “triple‐C”) locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head‐to‐tail by using the peptiligase variant omniligase‐1 (CEPS). Subsequent ligation–cyclization with tetravalent (T41/2) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF‐T41/2) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero‐bifunctional variants (UF/FU‐T41/2), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the “bifunctional tetracyclic peptide” inhibitor concept.