Scanning Ion-Conductance Microscopy for Studying ß-Amyloid Aggregate Formation on Living Cell Surfaces.

ß-Amyloid aggregation on living cell surfaces is described as responsible for the neurotoxicity associated with different neurodegenerative diseases. It is suggested that the aggregation of ß-amyloid (Aß) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss, etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules and consequently alterations of cell mechanical properties. The effect of amyloid oligomers on cells' Young's modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on the local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Young's modulus mapping, and confocal imaging of Aß aggregate formation on living cell surfaces. However, it is well-known that the cytoskeleton state is highly connected to the intracellular level of reactive oxygen species (ROS). The effect of Aß leads to the induction of oxidative stress, actin polymerization, and stress fiber formation. We measured the reactive oxygen species levels inside single cells using platinum nanoelectrodes to demonstrate the connection of ROS and Young's modulus of cells. SICM can be successfully applied to studying the cytotoxicity mechanisms of Aß aggregates on living cell surfaces.

Mass spectrometric studies of the variety of beta-amyloid proteoforms in Alzheimer's disease.

This review covers the results of the application of mass spectrometric (MS) techniques to study the diversity of beta-amyloid (Aß) peptides in human samples. Since Aß is an important hallmark of Alzheimer's disease (AD), which is a socially significant neurodegenerative disorder of the elderly worldwide, analysis of its endogenous variations is of particular importance for elucidating the pathogenesis of AD, predicting increased risks of the disease onset, and developing effective therapy. MS approaches have no alternative for the study of complex samples, including a wide variety of Aß proteoforms, differing in length and modifications. Approaches based on matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography with electrospray ionization tandem MS are most common in Aß studies. However, Aß forms with isomerized and/or racemized Asp and Ser residues require the use of special methods for separation and extra sensitive and selective methods for detection. Overall, this review summarizes current knowledge of Aß species found in human brain, cerebrospinal fluid, and blood plasma; focuses on application of different MS approaches for Aß studies; and considers the potential of MS techniques for further studies of Aß-peptides.

Role of Interaction between Zinc and Amyloid Beta in Pathogenesis of Alzheimer's Disease.

Progression of Alzheimer's disease is accompanied by the appearance of extracellular deposits in the brain tissues of patients with characteristic supramolecular morphology (amyloid plaques) the main components of which are ß-amyloid isoforms (Aß) and biometal ions (zinc, copper, iron). For nearly 40 years and up to the present time, the vast majority of experimental data indicate critical role of formation and accumulation of amyloid plaques (cerebral amyloidogenesis) in pathogenesis of Alzheimer's disease, however, nature of the molecular agents that initiate cerebral amyloidogenesis, as well as causes of aggregation of the native Aß molecules in vivo remained unknown for a long time. This review discusses the current level of fundamental knowledge about the molecular mechanisms of interactions of zinc ions with a number of Aß isoforms present in amyloid plaques of the patients with Alzheimer's disease, and also shows how this knowledge made it possible to identify driving forces of the cerebral amyloidogenesis in Alzheimer's disease and made it possible to determine fundamentally new biomarkers and drug targets as part of development of innovative strategy for diagnosis and treatment of Alzheimer's disease.

Electrochemical Analysis in Studying ß-Amyloid Aggregation.

ß-amyloid (Aß) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aß aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aß aggregation have intensively being investigated using synthetic Aß peptides by methods based on monitoring of aggregates, including determination of their size and structure. In this review, an orthogonal approach to the study of Aß aggregation is considered, which relies on electrochemical registration of the loss of peptide monomers. Electrochemical analysis of Aß (by voltammetry and amperometric flow injection analysis) is based on registration of the oxidation signal of electroactive amino acid residues of the peptide on an electrode surface. The Aß oxidation signal disappears, when the peptide is included in the aggregate. The advantages and disadvantages of electrochemical analysis for the study of spontaneous and metal-induced aggregation of Aß, comparative analysis of various peptide isoforms, and study of the process of complexation of metal ions with the metal-binding domain of Aß are discussed. It is concluded that the combined use of the electrochemical method and the methods based on detection of Aß aggregates makes it possible to obtain more complete information about the mechanisms of peptide aggregation.

Switching On/Off Amyloid Plaque Formation in Transgenic Animal Models of Alzheimer's Disease.

A hallmark of Alzheimer's disease (AD) are the proteinaceous aggregates formed by the amyloid-beta peptide (Aß) that is deposited inside the brain as amyloid plaques. The accumulation of aggregated Aß may initiate or enhance pathologic processes in AD. According to the amyloid hypothesis, any agent that has the capability to inhibit Aß aggregation and/or destroy amyloid plaques represents a potential disease-modifying drug. In 2023, a humanized IgG1 monoclonal antibody (lecanemab) against the Aß-soluble protofibrils was approved by the US FDA for AD therapy, thus providing compelling support to the amyloid hypothesis. To acquire a deeper insight on the in vivo Aß aggregation, various animal models, including aged herbivores and carnivores, non-human primates, transgenic rodents, fish and worms were widely exploited. This review is based on the recent data obtained using transgenic animal AD models and presents experimental verification of the critical role in Aß aggregation seeding of the interactions between zinc ions, Aß with the isomerized Asp7 (isoD7-Aß) and the α4ß2 nicotinic acetylcholine receptor.

Molecular Mechanism of Zinc-Dependent Oligomerization of Alzheimer's Amyloid-ß with Taiwan (D7H) Mutation.

Amyloid-ß (Aß) is a peptide formed by 39-43 amino acids, heterogenous by the length of its C-terminus. Aß constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer's disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aß seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aß isoforms, among which the most common is Aß with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aß aging. Aß molecules with isoD7 (isoD7-Aß) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aß. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aß is based on the rigidly structured segment 11-EVHH-14, located in the Aß metal binding domain (Aß16). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aß with Taiwan (D7H) mutation (D7H-Aß). In this study, the D7H-Aß metal binding domain (D7H-Aß16) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aß oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aß, as well as isoD7-Aß in combination with two Aß molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aß species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.

Tetrapeptide Ac-HAEE-NH2 Protects α4ß2 nAChR from Inhibition by Aß.

The cholinergic deficit in Alzheimer's disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aß peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the 11EVHH14 site in Aß peptide mediates its interaction with α4ß2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aß-α4ß2 nAChR complex is based on the interaction of 11EVHH14 with its charge-complementary counterpart in α4ß2 nAChR. Indeed, we discovered a 35HAEE38 site in α4ß2 nAChR, which is charge-complementary to 11EVHH14, and molecular modeling showed that a stable Aß42-α4ß2 nAChR complex could be formed via the 11EVHH14:35HAEE38 interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH2 can bind to Aß via 11EVHH14 site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH2 tetrapeptide completely abolishes the Aß42-induced inhibition of α4ß2 nAChR. Thus, we suggest that 35HAEE38 is a potential binding site for Aß on α4ß2 nAChR and Ac-HAEE-NH2 tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4ß2 nAChR-dependent cholinergic dysfunction in AD.

Application of MALDI-TOF/TOF-MS for relative quantitation of α- and ß-Asp7 isoforms of amyloid-ß peptide.

It is known that aspartic acid isomerization process plays a role in aging processes and may be used as a marker for aging of natural materials. As for Alzheimer's disease, the most abundant modification in the peptide profile is the aspartate isomerization of amyloid-ß. Liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry-based approaches with Collision Induced Dissociation (CID) or Electron Capture Dissociation (ECD) fragmentation provide a good and precise method for the relative quantitation of iso- to normal amyloid-ß peptides but require additional time consuming steps. In this study, MALDI-TOF/TOF-matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MS) method was developed as a high-throughput approach for the relative quantitation of the isomerized form of the amyloid-ß peptide.

A Binuclear Zinc Interaction Fold Discovered in the Homodimer of Alzheimer's Amyloid-ß Fragment with Taiwanese Mutation D7H.

Zinc-induced oligomerization of amyloid-ß peptide (Aß) produces potentially pathogenic agents of Alzheimer's disease. Mutations and modifications in the metal binding domain 1-16 of Aß peptide crucially affect its zinc-induced oligomerization by changing intermolecular zinc mediated interface. The 3D structure of this interface appearing in a range of Aß species is a prospective drug target for disease modifying therapy. Using NMR spectroscopy, EXAFS spectroscopy, mass spectrometry, and isothermal titration calorimetry the interaction of zinc ions with Aß fragments 1-7 and 1-10 carrying familial Taiwanese mutation D7H was studied. Zinc ions induce formation of a stable homodimer formed by the two peptide chains fastened by two zinc ions and stacking interactions of imidazole rings. A binuclear zinc interaction fold in the dimer structure was discovered. It can be used for designing zinc-regulated proteins and zinc-mediated self-assembling peptides.

The effects of endogenous non-peptide molecule isatin and hydrogen peroxide on proteomic profiling of rat brain amyloid-ß binding proteins: relevance to Alzheimer's disease?

The amyloid-ß peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-ß accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-ß binding proteins and 0.1 mM isatin decreased the number of identified amyloid-ß binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-ß binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-ß oligomers described in the literature for some isatin derivatives.

Zn-dependent ß-amyloid Aggregation and its Reversal by the Tetrapeptide HAEE.

The pathogenesis of Alzheimer's disease (AD) is associated with the formation of cerebral amyloid plaques, the main components of which are the modified Aß molecules as well as the metal ions. Aß isomerized at Asp7 residue (isoD7-Aß) is the most abundant isoform in amyloid plaques. We hypothesized that the pathogenic effect of isoD7-Aß is due to the formation of zinc-dependent oligomers, and that this interaction can be disrupted by the rationally designed tetrapeptide (HAEE). Here, we utilized surface plasmon resonance, nuclear magnetic resonance, and molecular dynamics simulation to demonstrate Zn2+-dependent oligomerization of isoD7-Aß and the formation of a stable isoD7-Aß:Zn2+:HAEE complex incapable of forming oligomers. To demonstrate the physiological importance of zinc-dependent isoD7-Aß oligomerization and the ability of HAEE to interfere with this process at the organismal level, we employed transgenic nematodes overexpressing human Aß. We show that the presence of isoD7-Aß in the medium triggers extensive amyloidosis that occurs in a Zn2+-dependent manner, enhances paralysis, and shortens the animals' lifespan. Exogenous HAEE completely reverses these pathological effects of isoD7-Aß. We conclude that the synergistic action of isoD7-Aß and Zn2+ promotes Aß aggregation and that the selected small molecules capable of interrupting this process, such as HAEE, can potentially serve as anti-amyloid therapeutics.

NMR solution structure of rat aß(1-16): toward understanding the mechanism of rats' resistance to Alzheimer's disease.

In an attempt to reveal the mechanism of rats' resistance to Alzheimer's disease, we determined the structure of the metal-binding domain 1-16 of rat ß-amyloid (rat Aß(1-16)) in solution in the absence and presence of zinc ions. A zinc-induced dimerization of the domain was detected. The zinc coordination site was found to involve residues His-6 and His-14 of both peptide chains. We used experimental restraints obtained from analyses of NMR and isothermal titration calorimetry data to perform structure calculations. The calculations employed an explicit water environment and a simulated annealing molecular-dynamics protocol followed by quantum-mechanical/molecular-mechanical optimization. We found that the C-tails of the two polypeptide chains of the rat Aß(1-16) dimer are oriented in opposite directions to each other, which hinders the assembly of rat Aß dimers into oligomeric aggregates. Thus, the differences in the structure of zinc-binding sites of human and rat Aß(1-16), their ability to form regular cross-monomer bonds, and the orientation of their hydrophobic C-tails could be responsible for the resistance of rats to Alzheimer's disease.

Development of Peptide Biopharmaceuticals in Russia.

Peptides are low-molecular-weight substances that participate in numerous important physiological functions, such as human growth and development, stress, regulation of the emotional state, sexual behavior, and immune responses. Their mechanisms of action are based on receptor-ligand interactions, which result in highly selective effects. These properties and low toxicity enable them to be considered potent drugs. Peptide preparations became possible at the beginning of the 20th century after a method was developed for selectively synthesizing peptides; however, after synthesis of the first peptide drugs, several issues related to increasing the stability, bioavailability, half-life, and ability to move across cell membranes remain unresolved. Here, we briefly review the history of peptide production and development in the biochemical industry and outline potential areas of peptide biopharmaceutical applications and modern approaches for creating pharmaceuticals based on synthetic peptides and their analogs. We also focus on original peptide drugs and the approaches used for their development by the Russian Federation.

Capabilities of MS for analytical quantitative determination of the ratio of α- and ßAsp7 isoforms of the amyloid-ß peptide in binary mixtures.

There is strong evidence that the amyloid-ß peptide (Aß) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), a lethal neurodegenerative disorder of the elderly. During pathology development, the peptide as well as its various chemically modified isoforms is accumulated in specific brain tissues as characteristic proteinaceous deposits, the so-called amyloid plaques, which are the pathomorphological mark of AD, although the level of Αß in the blood is the same for healthy individuals and for AD patients. Earlier, it has been shown that isomerization of aspartate 7, the most abundant post-translational modification of the Αß peptide, is tightly involved in a set of molecular processes associated with AD progression. Therefore, the isoAsp 7-containing Αß isomer (isoAß) is assumed to be a potential biomarker of AD that can be identified in the blood. Here, we present an analytical mass spectrometric method for quantitative determination of the ratio of normal and isomerized Αß fragments 1-16 in their binary mixtures, and all analytical capabilities, such as accuracy, detection limits, and sensitivity of the presented method, are determined and thoroughly discussed. On the basis of this method, an analytical approach for quantitative determination of this modification in the blood will be developed in further studies.

Pharmacokinetics and Molecular Modeling Indicate nAChRα4-Derived Peptide HAEE Goes through the Blood-Brain Barrier.

One of the treatment strategies for Alzheimer's disease (AD) is based on the use of pharmacological agents capable of binding to beta-amyloid (Aß) and blocking its aggregation in the brain. Previously, we found that intravenous administration of the synthetic tetrapeptide Acetyl-His-Ala-Glu-Glu-Amide (HAEE), which is an analogue of the 35-38 region of the α4 subunit of α4ß2 nicotinic acetylcholine receptor and specifically binds to the 11-14 site of Aß, reduced the development of cerebral amyloidogenesis in a mouse model of AD. In the current study on three types of laboratory animals, we determined the biodistribution and tissue localization patterns of HAEE peptide after single intravenous bolus administration. The pharmacokinetic parameters of HAEE were established using uniformly tritium-labeled HAEE. Pharmacokinetic data provided evidence that HAEE goes through the blood-brain barrier. Based on molecular modeling, a role of LRP1 in receptor-mediated transcytosis of HAEE was proposed. Altogether, the results obtained indicate that the anti-amyloid effect of HAEE, previously found in a mouse model of AD, most likely occurs due to its interaction with Aß species directly in the brain.

Extracellular GAPDH Promotes Alzheimer Disease Progression by Enhancing Amyloid-ß Aggregation and Cytotoxicity.

Neuronal cell death at late stages of Alzheimer's disease (AD) causes the release of cytosolic proteins. One of the most abundant such proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forms stable aggregates with extracellular amyloid-ß (Aß). We detect these aggregates in cerebrospinal fluid (CSF) from AD patients at levels directly proportional to the progressive stages of AD. We found that GAPDH forms a covalent bond with Q15 of Aß that is mediated by transglutaminase (tTG). The Q15A substitution weakens the interaction between Aß and GAPDH and reduces Aß-GAPDH cytotoxicity. Lentivirus-driven GAPDH overexpression in two AD animal models increased the level of apoptosis of hippocampal cells, neural degeneration, and cognitive dysfunction. In contrast, in vivo knockdown of GAPDH reversed these pathogenic abnormalities suggesting a pivotal role of GAPDH in Aß-stimulated neurodegeneration. CSF from animals with enhanced GAPDH expression demonstrates increased cytotoxicity in vitro. Furthermore, RX-624, a specific GAPDH small molecular ligand reduced accumulation of Aß aggregates and reversed memory deficit in AD transgenic mice. These findings argue that extracellular GAPDH compromises Aß clearance and accelerates neurodegeneration, and, thus, is a promising pharmacological target for AD.

Minimal Zn(2+) binding site of amyloid-ß.

Zinc-induced aggregation of amyloid-ß peptide (Aß) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aß region 6-14 as the minimal Zn(2+) binding site wherein the ion is coordinated by His(6), Glu(11), His(13), and His(14). With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11-14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn(2+)-induced aggregation of Aß.

Aberrant interactions between amyloid-beta and alpha5 laminins as possible driver of neuronal disfunction in Alzheimer's disease.

It has been widely accepted that laminins are involved in pathogenesis of Alzheimer's disease (AD). Amyloid plaques in AD patients are associated with immunostaining using antibodies raised against laminin-111, and laminin-111 has been shown to prevent aggregation of amyloid peptides. Although numerous articles describe small peptides from laminin-111 that are capable to disaggregate amyloid buildups and reduce neurotoxicity in in vitro and in vivo models, there is no approved laminin-111-based therapeutic approaches for treatment of AD. Also, it has been shown that immunoreactivity to laminin-111 appears late in development of cerebral amyloidosis. Based on the published data, we hypothesize that aberrant interaction between amyloid-beta and α5-laminins such as laminin-511 prevents the necessary laminin signaling into neurons leading to neurodegeneration and contributing to the early development of AD. Laminin-511 is the key extracellular protein that protects neurons from anoikis, inhibits excitoxicity and provides signaling that stabilizes dendritic spines and synapses in the developed brain. Absence of the signaling from laminin-511 leads to behavioral defects in mice. Laminin-511 and hippocampal neurons are in direct contact and accumulation of amyloid-beta that has been shown to avidly bind laminin-511 may physically decouple the interaction between α5-laminins and the neuronal membrane receptors inhibiting the signaling. Under this hypothesis, protein domains and peptides from laminin α5 chain may have a therapeutic potential in treatment of AD and the appearance of laminin-111 in the amyloid plaques is simply a consequence of the disease.

The English (H6R) Mutation of the Alzheimer's Disease Amyloid-ß Peptide Modulates Its Zinc-Induced Aggregation.

The coordination of zinc ions by histidine residues of amyloid-beta peptide (Aß) plays a critical role in the zinc-induced Aß aggregation implicated in Alzheimer's disease (AD) pathogenesis. The histidine to arginine substitution at position 6 of the Aß sequence (H6R, English mutation) leads to an early onset of AD. Herein, we studied the effects of zinc ions on the aggregation of the Aß42 peptide and its isoform carrying the H6R mutation (H6R-Aß42) by circular dichroism spectroscopy, dynamic light scattering, turbidimetric and sedimentation methods, and bis-ANS and thioflavin T fluorescence assays. Zinc ions triggered the occurrence of amorphous aggregates for both Aß42 and H6R-Aß42 peptides but with distinct optical properties. The structural difference of the formed Aß42 and H6R-Aß42 zinc-induced amorphous aggregates was also supported by the results of the bis-ANS assay. Moreover, while the Aß42 peptide demonstrated an increase in the random coil and ß-sheet content upon complexing with zinc ions, the H6R-Aß42 peptide showed no appreciable structural changes under the same conditions. These observations were ascribed to the impact of H6R mutation on a mode of zinc/peptide binding. The presented findings further advance the understanding of the pathological role of the H6R mutation and the role of H6 residue in the zinc-induced Aß aggregation.

Evaluation of MALDI-TOF/TOF Mass Spectrometry Approach for Quantitative Determination of Aspartate Residue Isomerization in the Amyloid-ß Peptide.

Immunoprecipitation (IP) combined with MALDI-TOF mass spectrometry is a powerful instrument for peptide and protein identification in biological samples. In this study, the analytical capabilities of MALDI-TOF/TOF mass spectrometry for relative quantitation of isoAsp7 in Aß(1-42) and Aß(1-16) were investigated. The possibility of quantitative determination of isoAsp7 in Aß(1-42) with the detection limit as low as 2 pmol has been demonstrated. The same approach was applied for a shorter peptide Aß(1-16) and resulted in enhanced accuracy (± 3.2%), and lower detection limit (50 fmol). Pilot experiments with artificial cerebrospinal fluid and mouse brain tissue were performed and showed that the proposed IP-MALDI-TOF/TOF approach could be applied for measuring isoAß content in biological fluids and tissues. Additionally, it was shown that 6E10 anti-amyloid antibodies might affect the accuracy of the amyloid-ß quantitation in the presence of the isomerized peptide.