C-terminal Peptide Research Articles

Triethyl phosphate (TEP) – dimethylsulfoxide (DMSO), as a green solvent mixture for solid-phase peptide synthesis (SPPS)

ABSTRACT The free use of DMF has recently been restricted since December 2023 in Europe because of its health hazards. Over time, these restrictions will extend to the USA and other countries. As DMF is the solvent of choice for solid-phase peptide synthesis (SPPS), its replacement has become an important issue. So far, there is not a solvent alone that shows possible replacement of DMF, consequently, several mixtures have been proposed. Herein, the triethyl phosphate (TEP) is proposed as an excellent alternative to EtOAc/BuOAc for use in combination with DMSO for SPPS at rt. TEP has shown broad application as a flame retardant in the plastics manufacturing industry due to its negligible level of toxicity, non-carcinogenicity, non-mutagenicity, and non-bioaccumulative properties. The performance of TEP-DMSO (3:1) in SPPS has been demonstrated by the synthesis of three medium-sized peptides of interest (Angiotensin, Afamelanotide, and the C-terminal peptide of Tirzepatide) with excellent purity at rt. Furthermore, TEP-DMSO (3:1) results in slightly less racemization than DMF in peptides containing Cys/His. Finally, it has been demonstrated that Ramage resin is more secure to use than Rink-amide resin because it does not break out during the final global deprotection step, avoiding the formation of linker-related side-products.

Teneurin C-Terminal Associated Peptide (TCAP)-1 Attenuates Restraint Stress-Induced Corticosterone Increases in Male Mice and Rats.

Hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis response can result in anxiety and other neuropsychiatric disorders and effective therapeutics are needed to mitigate this maladaptive response. Here we examined the effects of Teneurin C-terminal Associated Peptide (TCAP)-1, a peptide known to inhibit corticotropin releasing factor (CRF)-mediated stress, on the physiological expression of stress, and whether the effects of TCAP-1 were dependent on the route of administration. We first examined whether subcutaneous administration of TCAP-1 influenced tube restraint stress-induced corticosterone (CORT) increases in both male mice and rats. Using a similar model, we further examined the efficacy and time course of intranasal TCAP-1. Results showed that subcutaneous TCAP-1 administration attenuated the expression of the physiological manifestation of stress in male mice and rats, and that intranasal TCAP-1 delivered prophylactically is effective at attenuating stress-induced CORT increases in male rats. These data indicate that TCAP-1 delivered though non-invasive routes of administration could have potential as a clinically relevant anxiolytic.

Twisted Amide-Mediated Peptide Synthesis.

A robust, practical, and sustainable isomerization-suppressed peptide bond formation via acyl sulfonamide, a twisted amide, is disclosed. Tosyl isocyanate and pentafluorobenzyl bromide were applied in combination to activate the peptide C-terminus, which then reacted with an amine to yield an elongated peptide with high stereochemical purity. Careful analysis of NMR spectra of the active intermediate revealed the presence of an intramolecular hydrogen bond, suggesting that the hydrogen bond suppressed Cα-epimerization during amidation. The isomerization suppression by the intramolecular hydrogen bond is expected to be effective even under high dilution conditions, making the present method a powerful tool for the synthesis of complex macrocyclic peptides. In addition to peptide synthesis, the developed synthetic entry to twisted amides can be applied to the investigation of transition metal-catalyzed N-C bond activation. Moreover, the application to the N-C bond activation returned insight into peptide synthesis, leading to the use of sulfonamide as a protecting group of carboxylic acid that can be orthogonally removed in the presence of other conventional protecting groups.

Integrated computational characterization of valosin-containing protein double-psi β-barrel domain: Insights into structural stability, binding mechanisms, and evolutionary significance.

Valosin-containing protein (VCP) plays a crucial role in various cellular processes, yet the molecular mechanisms and structural dynamics of its double-psi β-barrel (DPBB) domain, particularly in human, remain insufficiently explored. While previous studies have characterized the VCP_DPBB domain in other organisms, such as thermoplasma acidophilum and methanopyrus kandleri, its evolutionary conservation, binding potential, and stability in human require further investigation. To address this gap, we first employed all-atom molecular dynamics (AAMD) simulations to examine the structural dynamics of the human VCP_DPBB domain. We also assessed its amino acid interaction energies, stability, folding enthalpy, evolutionary conservation, solubility, and crystallizability using various computational frameworks. Additionally, to uncover the plausible biological function, protein-peptide docking was performed to evaluate the interactions between the DPBB domain and the C-terminal gp78 peptide of the E3 ubiquitin ligase. Further, AAMD and coarse-grained molecular dynamics (CGMD) simulations explored the binding preferences, fluctuations, and stability of human VCP_DPBB-gp78 complexes. Our findings indicate that, while thermoplasma acidophilum VCP_DPBB-gp78 showed stronger initial binding, the human VCP_DPBB-gp78 complex exhibited superior stability, binding affinity, and more stabilizing interactions. This integrated analysis provides valuable insights into the evolutionary significance and functionality of the DPBB domain, with potential therapeutic implications for VCP-related diseases.

Bioluminescence assay of lysine deacylase sirtuin activity

Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine toward maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple conditions, including cancer. Measuring sirtuins' activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed "SIRTify", a high-sensitivity assay for measuring sirtuin activity invitro and invivo. SIRTify is based on a split-version of the NanoLuc luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine-acylations and confirm the effects of sirtuin modulators. SIRTify quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.

Antibacterial, Antibiofilm, and Anti-inflammatory Effects of a Novel Thrombin-Derived Peptide in Sepsis Models: Insights into Underlying Mechanisms.

We developed two short helical antimicrobial peptides, HVF18-a3 and its d-enantiomer, HVF18-a3-d, derived from the thrombin C-terminal peptide HVF18. These peptides exhibit potent antimicrobial activity against various bacteria by compromising both the outer and inner membranes, with low hemolytic activity. They are stable in the presence of physiological salts and human serum, exhibiting a low potential for developing drug resistance and excellent antibiofilm activity against Gram-negative bacteria. HVF18-a3-d also neutralized lipopolysaccharide (LPS) through direct binding interactions and suppressed the production of inflammatory cytokines through the inflammatory signaling pathway mediated by Toll-like receptor 4 in RAW264.7 cells stimulated with LPS. Both pre- and post-treatment with HVF18-a3-d significantly protected mice against fatal septic shock induced by carbapenem resistant Acinetobacter baumannii. These findings suggest HVF18-a3 and HVF18-a3-d are promising candidates for developing antibiotics against Gram-negative sepsis.

An Enabling Peptide Ligation Induced by Thiol-Salicylaldehyde Ester for Chemical Protein Synthesis.

Chemical protein synthesis by amide-forming ligation of two unprotected peptide segments offers an effective strategy for the preparation of protein derivatives that are not accessible through bioengineering approaches. Herein, an unprecedented chemical ligation between peptides with C-terminal 2-mercaptobenzaldehyde (thiol-salicylaldehyde, TSAL) esters and peptides bearing N-terminal cysteine/penicillamine is reported. Reactive peptide TSAL esters can be obtained from peptide hydrazides in an operationally simple and highly effective manner. This chemoselective peptide ligation enables the rapid production of N,S-benzylidene acetal intermediates, which can readily be converted into native amide bonds even at sterically hindered junctions. In addition, the current method can be applied compatibly in concert with other types of ligations and subsequent desulfurization chemistry, thereby facilitating convergent protein synthesis. The effectiveness of this new method is also showcased by the total synthesis of proteins ubiquitin and hyalomin-3 (Hyal-3), the efficient synthesis of protein ubiquitin-fold modifier 1 (UFM1) via a C-to-N sequential TSAL ester-induced ligation strategy, and the chemical synthesis of protein Mtb CM through a combined strategy of Ser/Thr ligation and TSAL ester-induced ligations.

α2δ-2 regulates synaptic GluK1 kainate receptors in Purkinje cells and motor coordination.

Gabapentin and pregabalin are inhibitory ligands of both α2δ-1 and α2δ-2 proteins (also known as subunits of voltage-activated Ca2+ channels) and are commonly prescribed for the treatment of neuropathic pain and epilepsy. However, these drugs can cause gait disorders and ataxia through unknown mechanisms. α2δ-2 and GluK1, a glutamate-gated kainate receptor subtype, are coexpressed in cerebellar Purkinje cells. In this study, we used a heterologous expression system and Purkinje cells to investigate the potential role of α2δ-2 in regulating GluK1-containing kainate receptor activity. Whole-cell patch clamp recordings showed that α2δ-2 coexpression augmented GluK1, but not GluK2, currents in HEK293 cells, and pregabalin abolished this augmentation. Pregabalin lost its inhibitory effect on GluK1 currents in HEK293 cells expressing both GluK1 and the α2δ-2(R282A) mutant. Blocking GluK1-containing receptors with UBP310 substantially reduced the amplitude of excitatory postsynaptic currents at parallel fiber-Purkinje cell synapses in mice. Also, pregabalin markedly attenuated the amplitude of excitatory postsynaptic currents and currents elicited by ATPA, a selective GluK1 receptor agonist, in Purkinje cells in Cacna2d1 knockout mice. Coimmunoprecipitation assays indicated that α2δ-2, but not α2δ-1, formed a protein complex with GluK1 in cerebellar tissues and HEK293 cells through its C terminus. Furthermore, α2δ-2 coexpression potentiated surface expression of GluK1 proteins in HEK293 cells, whereas pregabalin reduced GluK1 proteins in cerebellar synaptosomes. Disrupting α2δ-2-GluK1 interactions using α2δ-2 C-terminus peptide abrogated the potentiating effect of α2δ-2 on GluK1 currents and attenuated the amplitude of GluK1-mediated excitatory postsynaptic currents in Purkinje cells. However, neither pregabalin nor α2δ-2 C-terminus peptide had significant effect on P/Q-type currents in HEK293 cells. Additionally, CRISPR/Cas9-induced conditional knockdown of Cacna2d2 or Grik1 in Purkinje cells, as well as microinjection of α2δ-2 C-terminus peptide or UBP310 into the cerebellum, substantially impaired beam walking and rotarod performance in mice. Our study reveals that α2δ-2 directly interacts with GluK1 independently of its conventional role as a voltage-activated Ca2+ channel subunit. α2δ-2 regulates motor coordination by promoting synaptic expression and activity in GluK1-containing kainate receptors in Purkinje cells.

Multipartite Fluorogenic Sensors for Monitoring Tyrosine Phosphatase Activity.

Fluorogenic substrates are essential tools for studying the activity of many enzymes including the protein tyrosine phosphatases (PTPs). Here, we have taken the first step toward the development of genetically encodable sensors for PTP activity using fluorescent and fluorogen-activating proteins. The Fluorescence-Activating and absorption Shifting Tag (FAST) is a small protein that becomes fluorescent upon binding to a small molecule dye. We demonstrate that FAST protein can be used as a sensor for PTP-mediated dephosphorylation of phosphorylated dye molecules. Phosphorylated 4-hydroxybenzylidene rhodanine (pHBR) is not able to bind to the FAST protein and induce fluorescence, but provides a sensitive assay for PTP activity, readily detecting 100 pM concentrations of PTP1B in the presence of FAST with a kcat value of 19±1 s-1 and a KM value of 93±3 μM. In addition, while phosphorylation of the C-terminal peptide of split GFP does not result in appreciable change in fluorescence of the reconstituted protein, phosphorylation of the C-terminal peptide of the split FAST protein abrogates fluorescence. Upon PTP-mediated dephosphorylation of the C-terminal peptide, the ability of the N- and C-terminal components to form a fluorescent complex with the small molecule dye is restored, leading to fluorescence.

Enhancing De Novo Protein Sequencing through the C-Terminal Labeling Strategy: Resolving Isobaric Ambiguities by Electron-Transfer/Higher Energy Collision Dissociation (EThcD).

De novo protein sequencing via a bottom-up approach requires various proteases to produce overlapping peptides. However, peptides generated by proteases other than trypsin, LysC, and ArgC often yield C-terminal fragments with suboptimal ionization in positive mode mass spectrometry (MS). This study introduces a novel peptide labeling strategy that involves modifying peptides at the C-terminal and at the carboxyl groups of Aspartic and Glutamic acid with arginine methyl ester (R-met) to improve peptide fragmentation and resolve isobaric ambiguities encountered during sequencing. An amidation reaction is used with coupling reagents to conjugate R-met to the peptide's C-terminal end, introducing a functional group that enhances the detectability of C-terminal peptide fragment ions by mass spectrometry. Subsequently, selecting a charge state of +2 or higher can facilitate optimal fragmentation of the derivatized peptides using electron-transfer/higher energy collision dissociation (EThcD), thereby generating essential w-ions to resolve common isobaric ambiguities. Demonstrating this strategy across diverse protein types, including albumin and antibodies and using different proteases for digestion, highlights the unique characteristics of combining the proposed amidation reaction with the specific proteases tested.

Split MutT prevents the mutator phenotype of mutT-deficient Escherichia coli

BackgroundThe Escherichia coli MutT (NudA) protein catalyzes the hydrolysis of an oxidized form of dGTP, 8-oxo-7,8-dihydro-dGTP (8-hydroxy-dGTP), and the spontaneous mutation frequency is elevated in E. coli cells deficient in the mutT gene.ResultsA split MutT, comprising the N-terminal (residues 1–95) and C-terminal (residues 96–129) peptides, was designed based on the known tertiary structure and linker insertion mutagenesis experiments. The mutator phenotype was complemented when the two peptides were separately expressed in mutT E. coli cells.ConclusionsThese results indicated that this split MutT functions as a nucleotide pool sanitization enzyme in vivo.

8410 Lipid modification enhances the calcemic and bone building effects of injected PTH(1-14) and PTH(1-11) fragment peptides in mice

Abstract Disclosure: J. Höppner: None. H. Noda: None. A. Khatri: None. H. Jüppner: None. T.J. Gardella: None. PTH analogs with improved actions in vivo are of interest as potential treatment options for bone and mineral ion diseases such as hypoparathyroidism and osteoporosis. The efficacies in vivo of conventional PTH peptides are generally limited by rapid rates of clearance from the circulation (t1/2 = ∼ 5-30 minutes) and relatively short-dwell times on the receptor (PTH1R). This is especially true for small fragment analogs, such as M (modified)-PTH(1-14), and M-PTH(1-11), which have only been shown to be active in vitro. We explored whether PTH fragment peptide efficacy in vivo could be enhanced by addition of a lipid chain (e.g., a C15 palmitoyl chain (palm)) to either the peptide C-terminus or to the side chain of the amino acid residue at position 11 or 13. The C-tail lipid modifications were specifically designed to promote binding to serum albumin, and hence extend circulation half-life, while the lipid chains at sidechains of residue positions 11 and 13 were designed, based on structural models, to anchor the ligand to the receptor in situ -- i.e. by projection of the acyl chain between two adjacent transmembrane helices and into the surrounding plasma membrane -- to thereby extend receptor dwell time. Assessment of cAMP signaling potencies in HEK293/hPTH1R/glosensor cells revealed that both types of lipid modifications can preserve agonist potency, and that the lipid extensions at position 11 or 13 can indeed prolong receptor dwell times, as shown by extended durations of cAMP signaling after initial binding of Palm 11 or 13-modified M-PTH(1-14) and M-PTH(1-11) peptide fragment. Single subcutaneous injection of either Palm-13-M-PTH(1-14) or Palm-11-M-PTH(1-11) into mice resulted in prolonged increases in blood serum calcium levels, while 14 days of daily injection resulted in profound increases in bone mass, as measured by uCT, and marked increases in blood levels of the bone turnover markers CTX1 and P1NP, whereas non-lipidated M-PTH(1-14) control peptides were inactive in vivo. Peptide lipidation thus holds promise as a strategy to employ in the design of new PTH analogs with improved efficacies in vivo. Presentation: 6/1/2024

6616 Copeptin Use To Reduce Hospital Stay In A Complicated DI Case

Abstract Disclosure: S.S. Kowsika: None. H. Moran: None. Introduction: While there is literature describing the use of copeptin for the diagnosis of polyuria-polydipsia syndrome, its utilization remains very limited in many hospitals. We present a case where the patient, initially misdiagnosed with central diabetes insipidus (DI), was appropriately diagnosed with partial nephrogenic DI through the use of copeptin. Case: A 48-year old female with a history of cardiac arrest, anoxic brain injury and diabetes insidious (DI) presented to the emergency department with hypernatremia (Na 154 mmol/L). The patient had been initiated on desmopresin (DDAVP) at an outside facility prior to her current admission. Despite receiving high doses of DDAVP, her sodium levels remained elevated, prompting further investigation. DDAVP administration was discontinued. A water deprivation test was subsequently performed, followed by IV administration of 2 mcg of DDAVP, which resulted in an increase in urine osmolality from 191 to 331 mosm/kg, which was less than the expected 50% increase observed in central DI. Additionally, a baseline copeptin level was obtained and noted to be 31 pmol/L, confirming the diagnosis of partial nephrogenic DI. The patient was started on Amiloride, leading to normalization of sodium levels. Discussion: Many patients with polyuria-polydipsia syndrome are frequently misdiagnosed and receive incorrect treatment, potentially worsening their clinical condition. Copeptin (Carboxy-Terminal-Pro-vasopressin) is the C-terminal peptide of pro-vasopressin, co-secreted with arginine vasopressin (AVP). It has been proposed as a direct test for diagnosing polyuria-polydipsia syndrome. Baseline Copeptin testing without stimulation can accurately diagnose nephrogenic DI if elevated (>21.4, pmol/L). To differentiate between central DI and chronic polyuria, prior thirsting with hypertonic saline or glucagon stimulation is necessary, with stimulated copeptin <4.9 pmol/L diagnosing central DI. Despite its diagnostic potential, Copeptin is underutilized in many settings. In this case, using Copeptin to accurately diagnose our patient led to appropriate treatment and the resolution of hypernatremia. Presentation: 6/1/2024

Biosynthesis of peptide-nucleobase hybrids in ribosomal peptides.

The main biopolymers in nature are oligonucleotides and polypeptides. However, naturally occurring peptide-nucleobase hybrids are rare. Here we report the characterization of the founding member of a class of peptide-nucleobase hybrid natural products with a pyrimidone motif from a widely distributed ribosomally synthesized and post-translationally modified (RiPP) biosynthetic pathway. This pathway features two steps where a heteromeric RRE-YcaO-dehydrogenase complex catalyzes the formation of a six-membered pyrimidone ring from an asparagine residue on the precursor peptide, and an acyl esterase selectively recognizes this moiety to cleave the C-terminal follower peptide. Mechanistic studies reveal that the pyrimidone formation occurs in a substrate-assisted catalysis manner, requiring a His residue in the precursor to activate asparagine for heterocyclization. Our study expands the chemotypes of RiPP natural products and the catalytic scope of YcaO enzymes. This discovery opens avenues to create artificial biohybrid molecules that resemble both peptide and nucleobase, a modality of growing interest.

Photocatalytic Decarboxylative Allylation of α-Amino Acids and Peptides under Metal-Free Conditions.

We developed an organophotoredox catalytic system to facilitate the decarboxylative allylation coupling process concerning α-amino acids and related C-terminal carboxylate peptides using Morita-Baylis-Hillman adducts as allylic precursors. This metal-free method operates under mild conditions and is compatible with various amino acids. The versatility of this protocol, particularly in chemical biology research, has been preliminarily demonstrated through the ligation of bioactive peptide chains.

The growth-blocking peptide is a dual regulator of development and immunity with biocontrol potential in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Insect growth-blocking peptides (GBPs) are a family of cytokines found in several insect orders and are known for their roles in regulating development, paralysis, cell proliferation, and immune responses. Despite their diverse functions, the potential of GBPs as biocontrol targets against the pest Spodoptera frugiperda (Lepidoptera: Noctuidae) has not been fully explored. In this study, S. frugiperda GBP (SfGBP) was identified and functionally characterized. SfGBP is synthesized as a 146 amino acid proprotein with a 24 amino acid C-terminal active peptide (Glu123-Gly146). Predominant expression of SfGBP occurs in fourth to sixth instar larvae and in the larval fat body, with significant upregulation in response to pathogens and pathogen-associated molecular patterns. Injection of the synthetic active peptide into larvae induced growth retardation, delayed pupation, and increased survival against Beauveria bassiana infection. Conversely, RNA interference-mediated knockdown of SfGBP resulted in accelerated growth, earlier pupation, and decreased survival against B. bassiana infection. Further analysis revealed that SfGBP promoted SF9 cell proliferation and spreading, enhanced bacteriostatic activity of larval hemolymph, and directly inhibited germination of B. bassiana conidia. In addition, SfGBP enhanced humoral responses, such as upregulation of immunity-related genes and generation of reactive oxygen species, and cellular responses, such as nodulation, phagocytosis, and encapsulation. These results highlight the dual regulatory role of SfGBP in development and immune responses and establish it as a promising biocontrol target for the management of S. frugiperda.

Structural and biochemical analysis of highly similar HLA-B allotypes differentially associated with type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease involving T cell-mediated destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. CD8+ T cells, responding to beta cell peptides presented by class I major histocompatibility complex (MHC) molecules, are important effectors leading to beta cell elimination. Human leukocyte antigen (HLA) B∗39:06, B∗39:01, and B∗38:01 are closely related class I MHC allotypes that nonetheless show differential association with T1D. HLA-B∗39:06 is the most predisposing of all HLA class I molecules and is associated with early age at disease onset. B∗39:01 is also associated with susceptibility to T1D, but to a lesser extent, though differing from B∗39:06 by only two amino acids. HLA-B∗38:01, in contrast, is associated with protection from the disease. Upon identifying a peptide that binds to both HLA-B∗39:06 and B∗39:01, we determined the respective X-ray structures of the two allotypes presenting this peptide to 1.7Å resolution. The peptide residues available for T cell receptor contact and those serving as anchors were identified. Analysis of the F pocket of HLA-B∗39:06 and B∗39:01 provided an explanation for the distinct peptide C terminus preferences of the two allotypes. Structure-based modeling of the protective HLA-B∗38:01 suggested a potential reason for its peptide preferences and its reduced propensity to present 8-mer peptides compared to B∗39:06. Notably, the three allotypes showed differential binding to peptides derived from beta cell autoantigens. Taken together, our findings should facilitate identification of disease-relevant candidate T cell epitopes and structure-guided therapeutics to interfere with peptide binding.

The C-terminal self-binding helical peptide of human estrogen-related receptor γ can be druggably targeted by a novel class of rationally designed peptidic antagonists.

Orphan nuclear estrogen-related receptor γ (ERRγ) has been recognized as a potential therapeutic target for cancer, inflammation and metabolic disorder. The ERRγ contains a regulatory AF2 helical tail linked C-terminally to its ligand-binding domain (LBD), which is a self-binding peptide (SBP) and serves as molecular switch to dynamically regulate the receptor alternation between active and inactive states by binding to and unbinding from the AF2-binding site on ERRγ LBD surface, respectively. Traditional ERRγ modulators are all small-molecule chemical ligands that can be classified into agonists and inverse agonists in terms of their action mechanism; the agonists stabilize the AF2 in ABS site with an agonist conformation, while the inverse agonists lock the AF2 out of the site to largely abolish ERRγ transcriptional activity. Here, a class of ERRγ peptidic antagonists was described to compete with native AF2 for the ABS site, thus blocking the active state of AF2 binding to ERRγ LBD domain. Self-inhibitory peptide was derived from the SBP-covering AF2 region and we expected it can rebind potently to the ABS site by reducing its intrinsic disorder and entropy cost upon the rebinding. Hydrocarbon stapling was employed to do so, which employed an all-hydrocarbon bridge across the [i, i + 4]-anchor residue pair in the N-terminal, middle or C-terminal region of the self-inhibitory peptide. As might be expected, it is revealed that the stapled peptides are good binders of ERRγ LBD domain and can effectively compete with the native AF2 helical tail for ERRγ ABS site, which exhibit a basically similar binding mode with AF2 to the site and form diverse noncovalent interactions with the site, thus conferring stability and specificity to the domain-peptide complexes.

Teneurin C-terminal associated peptide (TCAP)-1 attenuates the development and expression of naloxone-precipitated morphine withdrawal in male Swiss Webster mice.

Corticotropin-releasing factor (CRF), the apical stress-inducing hormone, exacerbates stress and addictive behaviors. TCAP-1 is a peptide that directly inhibits both CRF-mediated stress and addiction-related behaviors; however, the direct action of TCAP-1 on morphine withdrawal-associated behaviors has not previously been examined. To determine whether TCAP-1 administration attenuates behavioral and physiological consequences of morphine withdrawal in mice. Mice were administered via subcutaneous route TCAP-1 either before or after initial morphine exposure, after which jumping behavior was quantified to assess the effects of TCAP-1 on naloxone-precipitated morphine withdrawal. As a comparison, mice were treated with nonpeptide CRF1 receptor antagonist CP-154,526. In one experiment, plasma corticosterone (CORT) was also measured as a physiological stress indicator. Pretreatment with TCAP-1 (10-250 nmol/kg) before morphine treatment significantly inhibited the development of naloxone-precipitated withdrawal. TCAP-1 (250-500 nmol/kg) treatment administered after morphine treatment attenuated the behavioral expression of naloxone-precipitated withdrawal. TCAP-1 (250 nmol/kg) treatment during morphine treatment was more effective than the optimal dosing of CP-154,526 (20mg/kg) at suppressing the behavioral expression of naloxone-precipitated withdrawal, despite similar reduction of withdrawal-induced plasma CORT level increases. These findings establish TCAP-1 as a potential therapeutic candidate for the prevention and treatment of morphine withdrawal.

Thrombin-derived C-terminal peptides bind and form aggregates with sulfated glycosaminoglycans

Glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) play crucial roles in inflammation and wound healing, serving as regulators of growth factors and pro-inflammatory mediators. In this study, we investigated the influence of heparin/HS on thrombin proteolysis and its interaction with the generated 11 kDa thrombin-derived C-terminal peptides (TCPs). Employing various biochemical and biophysical methods, we demonstrated that 11 kDa TCPs aggregate in the presence of GAGs, including heparin, heparan sulfate, and chondroitin sulfate-B. Circular dichroism analysis demonstrated that 11 kDa TCPs, in the presence of GAGs, adopt a β-sheet structure, a finding supported by thioflavin T1 (ThT) fluorescence measurements and visualization of 11 kDa TCP-heparin complexes using transmission electron microscopy (TEM). Furthermore, our investigations revealed a stronger binding affinity between 11 kDa TCPs and GAGs with higher sulfate group contents. Congruently, in silico simulations showed that interactions between 11 kDa TCPs and heparin/HS are predominantly electrostatic in nature. Collectively, our study suggests that 11 kDa TCPs have the capacity to aggregate in the presence of GAGs, shedding light on their potential roles in inflammation and wound healing.