Alvaro I. Herrera

The Central Kink Region of Fowlicidin-2, an α-Helical Host Defense Peptide, Is Critically Involved in Bacterial Killing and Endotoxin Neutralization

Fowlicidins are a group of newly identified chicken cathelicidin host defense peptides. We have shown that the putatively mature fowlicidin-2 of 31 amino acid residues possesses potent antibacterial and lipopolysaccharide (LPS)- neutralizing activities, but with a noticeable toxicity to mammalian cells. As a first step in exploring the structure-activity relationships of fowlicidin-2, in this study we determined its tertiary structure by nuclear magnetic resonance spectroscopy. Unlike the majority of cathelicidins, which are composed of a predominant α-helix with a short hinge sequence near the center, fowlicidin-2 consists of 2 well-defined α-helical segments (residues 6–12 and 23–27) connected by a long extensive kink (residues 13–20) induced by proline. To further investigate the functional significance of each of these structural components, several N- and C-terminal deletion analogs of fowlicidin-2 were synthesized and analyzed for their antibacterial, cytotoxic and LPS-neutralizing activities. Our results indicated that neither the N- nor C-terminal α-helix alone is sufficient to confer any function. Rather, fowlicidin-2(1–18) and fowlicidin-2(15–31), 2 α-helical segments with inclusion of the central cationic kink region, retained substantial capacities to kill bacteria and neutralize the LPS-induced proinflammatory response, relative to the parent peptide. More desirably, these 2 peptide analogs showed substantially reduced toxicity to human erythrocytes and epithelial cells, indicative of improved potential as antibacterial and antisepsis agents. To our knowledge, fowlicidin-2 is the first α-helical cathelicidin, with the central kink region shown to be critically important in killing bacteria and neutralizing LPS.

Thermally Induced Conformational Transitions in Nascent Branched Amphiphilic Peptide Capsules

Branched amphiphilic peptide capsules (BAPCs) are biocompatible, bilayer delimited polycationic nanospheres that spontaneously form at room temperature through the coassembly of two amphiphilic branched peptides: bis(FLIVI)-K-K4 and bis(FLIVIGSII)-K-K4. BAPCs are readily taken up by cells in culture, where they escape and/or evade the endocytic pathway and accumulate in the perinuclear region, persisting there without apparent degradation or extravasation. Drugs, small proteins, and solutes as well as α particle emitting radionuclides are stably encapsulated for extended periods of time. BAPC formation at room temperature proceeds via a fusogenic process and after 48 h a range of BAPCs sizes are observed, from 50 nm to a few microns in diameter. It was previously reported that cooling BAPCs from 25 to 4 °C and then back to 25 °C eliminated their fusogenic property. In this report, biophysical techniques reveal that BAPCs undergo thermosensitive conformational transitions as a function of both time and temperature and that the properties of BAPCs vary based on the temperature of assembly. The solvent dissociation properties of BAPCs were studied as well as the contributions of specific amino acid residues to the observed conformations. The roles of the potential stabilizing forces present within the bilayer that bestow the unusal stability of the BAPCs are discussed. Ultimately this study presents revised assembly protocols for preparing BAPCs with discrete sizes and solvent-induced extravasation properties.

Membrane Interacting Peptides: a Review.

Membrane interacting peptides of natural or synthetic origins serve a variety of biological purposes. They have been extensively studied for their involvement in immunity, diseases, and for their potential as medical therapeutics and research tools. In this review membrane interacting peptides are categorized into four groups according to their function: antimicrobial peptides, cell-penetrating peptides, channel forming peptides and amyloid peptides. A historical overview of the development, their functional mechanisms, and recent advances are presented for each of the groups. Considerable research is still devoted to this field of study and in this report a representative sample of the latest studies is presented. A set of common features among peptide groups emerges as the understanding of their action mechanisms increase. The possibility of a membrane centric general model for peptide-membrane interaction is also discussed. This review seeks to provide a unifying view of the field and promote the interaction between research groups working on peptides that have so far been studied as belonging to completely different fields.

Structural characterization of two pore-forming peptides: Consequences of introducing a C-terminal tryptophan

Synthetic channel‐forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among a series of 22‐residue peptides derived from the second transmembrane segment of the glycine receptor α1‐subunit (M2GlyR), p22‐S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one‐half maximal short circuit current is reduced to 45 ± 6 μM from that of 210 ± 70 μM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Toward obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22‐S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membrane‐anchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22‐S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C‐terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR‐derived peptides toward potential peptide‐based channel replacement therapy. Proteins 2010.

Backbone and side-chain 1H, 15N, and 13C resonance assignments of a novel Staphylococcal inhibitor of myeloperoxidase

The bacterium Staphylococcus aureus produces an array of anti-inflammatory molecules that prevent the innate immune system from recognizing it as a pathogen and clearing it from the host. In the acute phase of inflammation, our immune system relies on neutrophils to clear invading bacteria. Recently, novel classes of secreted proteins from S. aureus, including the Extracellular Adherence Protein (EAP) family (Stapels et al., Proc Natl Acad Sci USA 111:13187–13192, 2014) and the Staphylococcal Peroxidase Inhibitor (SPIN), (unpublished work) have been identified as highly selective inhibitors acting on Neutrophil Serine Proteases (NSPs) and myeloperoxidase (MPO) respectively. SPIN is a protein found only in Staphylococci, with no sequence homology to any known proteins. Solution NMR structural studies of SPIN are therefore expected to provide a deeper understanding of its interaction with MPO. In this study, we report the backbone and side-chain 1H, 15N, and 13C resonance assignments of SPIN. Furthermore, using the chemical shifts of these resonances, we predicted the secondary structure of SPIN in solution via the TALOS-N server. The assignment data has been deposited in the BMRB data bank under Accession No. 27069.

A Structurally Dynamic N-terminal Region Drives Function of the Staphylococcal Peroxidase Inhibitor (SPIN)

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal β-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a β-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal β-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN–MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins.

CHAPTER 15:Structure and Function of Stress-Responsive Peptides in Insects

Similar to innate immunity in vertebrates, insects rely on a variety of both humoral and cellular responses to defend them from pathogen invasion. Recently, a family of peptides has been identified in many insects including mosquitoes, that are activated or released during stress conditions, such as wounding, infection, ligation, crowdedness, and heat or cold shock. These peptides, expressed in the brain, midgut, fat body, and hemocytes play an important role in immunity and development of insects. These peptides are known as stress-response peptides. This chapter describes the structure, function, and characteristics concerning their known interactions with receptors.

Characterization of the ClC-1 Conductance at the Surface and Transverse Tubular System Membranes of Mammalian Skeletal Muscle Fibers

ClC-1 is believed to be primarily responsible for the resting conductance in mammalian skeletal muscle fibers. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltage-clamped enzymatically dissociated short fibers using a 2-microelectrode configuration and simultaneously recorded chloride currents (ICl), and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all, but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride in order to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl-dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from the TTS. Supported by NIH grants AR047664, AR041802, and AR054816.