Dimeric Peptide Research Articles

Lysine Acetylation Changes the Mechanism of Aβ25-35 Peptide Binding and Dimerization in the DMPC Bilayer.

The impact of Lys28 acetylation on Alzheimer's Aβ peptide binding to the lipid bilayer has not been previously studied, either experimentally or computationally. To probe this common post-translational modification, we performed all-atom replica exchange molecular dynamics simulations targeting binding and aggregation of acetylated acAβ25-35 peptide within the DMPC bilayer. Using the unmodified Aβ25-35 studied previously as a reference, our results can be summarized as follows. First, Lys28 acetylation strengthens the Aβ25-35 hydrophobic moment and consequently promotes the helical structure across the peptide extending it into the N-terminus. Second, because Lys28 acetylation disrupts electrostatic contact between Lys28 and lipid phosphate groups, it reduces the binding affinity of acAβ25-35 peptides to the DMPC bilayer. Accordingly, although acetylation preserves the bimodal binding featuring a preferred inserted state and a less probable surface bound state, it decreases the stability of the former. Third, acetylation promotes acAβ25-35 aggregation and eliminates monomers as thermodynamically viable species. More importantly, acAβ25-35 retains as the most thermodynamically stable the inserted dimer with unique head-to-tail helical aggregation interface. However, due to enhanced helix structure, this dimer state becomes less stable and is less likely to propagate into higher order aggregates. Thus, acetylation is predicted to facilitate the formation of low-molecular-weight oligomers. Other post-translational modifications, including phosphorylation and oxidation, reduce helical propensity and have divergent impact on aggregation. Consequently, acetylation, when considered in its totality, has distinct consequences on Aβ25-35 binding and aggregation in the lipid bilayer.

Amyloid Against Amyloid: Dimeric Amyloid Fragment Ameliorates Cognitive Impairments by Direct Clearance of Oligomers and Plaques

AbstractAmyloid‐β (Aβ) in the form of neurotoxic aggregates is regarded as the main pathological initiator and key therapeutic target of Alzheimer's disease. However, anti‐Aβ drug development has been impeded by the lack of a target needed for structure‐based drug design and low permeability of the blood–brain barrier (BBB). An attractive therapeutic strategy is the development of amyloid‐based anti‐Aβ peptidomimetics that exploit the self‐assembling nature of Aβ and penetrate the BBB. Herein, we designed a dimeric peptide drug candidate based on the N‐terminal fragment of Aβ, DAB, found to cross the BBB and solubilize Aβ oligomers and fibrils. Administration of DAB reduced amyloid burden in 5XFAD mice, and downregulated neuroinflammation and prevented memory impairment in the Y‐maze test. Peptide mapping assays and molecular docking studies were utilized to elucidate DAB‐Aβ interaction. To further understand the active regions of DAB, we assessed the dissociative activity of DAB with sequence modifications.

Amyloid Against Amyloid: Dimeric Amyloid Fragment Ameliorates Cognitive Impairments by Direct Clearance of Oligomers and Plaques.

Amyloid-β (Aβ) in the form of neurotoxic aggregates is regarded as the main pathological initiator and key therapeutic target of Alzheimer's disease. However, anti-Aβ drug development has been impeded by the lack of a target needed for structure-based drug design and low permeability of the blood-brain barrier (BBB). An attractive therapeutic strategy is the development of amyloid-based anti-Aβ peptidomimetics that exploit the self-assembling nature of Aβ and penetrate the BBB. Herein, we designed a dimeric peptide drug candidate based on the N-terminal fragment of Aβ, DAB, found to cross the BBB and solubilize Aβ oligomers and fibrils. Administration of DAB reduced amyloid burden in 5XFAD mice, and downregulated neuroinflammation and prevented memory impairment in the Y-maze test. Peptide mapping assays and molecular docking studies were utilized to elucidate DAB-Aβ interaction. To further understand the active regions of DAB, we assessed the dissociative activity of DAB with sequence modifications.

A snake venom-analog peptide that inhibits SARS-CoV-2 and papain-like protease displays antithrombotic activity in mice arterial thrombosis model, without interfering with bleeding time

Background(p-BthTX-I)2 K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required.ObjectivesTo determine whether (p-BthTX-I)2 K affects the hemostatic system.MethodsPlatelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I)2 K (5.0–434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5–20 mg kg− 1 Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I)2 K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I)2 K (4.0 mg/kg and 8.0 mg/kg) or saline administration.Results(p-BthTX-I)2 K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I)2 K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I)2 K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I)2 K did not prolong the bleeding time in the mouse model of arterial thrombosis.ConclusionThese results demonstrate the antithrombotic activity of the peptide (p-BthTX-I)2 K possibly by kallikrein inhibition, suggesting its strong biotechnological potential.

Prebiotic dimer and trimer peptide formation in gas-phase atmospheric nanoclusters of water.

Insight into the origin of prebiotic molecules is key to our understanding of how living systems evolved into the complex network of biological processes on Earth. By modelling diglycine and triglycine peptide formation in the prebiotic atmosphere, we provide a plausible pathway for peptide growth. By examining different transition states (TSs), we conclude that the formation of diglycine and triglycine in atmospheric nanoclusters of water in the prebiotic atmosphere kinetically favors peptide growth by an N-to-C synthesis of glycines through a trans conformation. Addition of water stabilizes the TS structures and lowers the Gibbs free activation energies. At temperatures that model the prebiotic atmosphere, the free energies of activation with a six water nanocluster as part of the TS are predicted to be 16 kcal mol-1 relative to the prereactive complex. Examination of the trans vs. cis six water transition states reveals that a homodromic water network that maximizes the acceptor/donor nature of the six waters is responsible for enhanced kinetic favorability of the trans N-to-C pathway. Compared to the non-hydrated trans TS, the trans six-water TS accelerates the reaction of diglycine and glycine to form triglycine by 13 orders of magnitude at 217 K. Nature uses the trans N-to-C pathway to synthesize proteins in the ribosome, and we note the similarities in hydrogen bond stabilization between the transition state for peptide synthesis in the ribosome and the transition states formed in nanoclusters of water in the same pathway. These results support the hypothesis that small oligomers formed in the prebiotic atmosphere and rained onto earth's surface.

Properties and Activity of Peptide Derivatives of ACE2 Cellular Receptor and Their Interaction with SARS-CoV-2 S Protein Receptor-Binding Domain

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture.

Near-infrared-dye labeled tumor vascular-targeted dimer GEBP11 peptide for image-guided surgery in gastric cancer.

Positive resection margins occur in about 2.8%-8.2% gastric cancer surgeries and is associated with poor prognosis. Intraoperative guidance using Nearinfrared (NIR) fluorescence imaging is a promising technique for tumor detection and margin assessment. The goal of this study was to develop a tumor-specific probe for real-time intraoperative NIR fluorescence imaging guidance. The tumor vascular homing peptide specific for gastric cancer, GEBP11, was conjugated with a near-infrared fluorophore, Cy5.5. The binding specificity of the GEBP11 probes to tumor vascular endothelial cells were confirmed by immunofluorescent staining. The ability of the probe to detect tumor lesions was evaluated in two xenograft models. An orthotopic gastric cancer xenograft model was used to evaluate the efficacy of the GEBP11 NIR probes in real-time surgical guidance. In vitro assay suggested that both mono and dimeric GEBP11 NIR probes could bind specifically to tumor vascular epithelial cells, with dimeric peptides showed better affinity. In tumor xenograft mice, live imaging suggested that comparing with free Cy5.5 probe, significantly stronger NIR signals could be detected at the tumor site at 24-48h after injection of mono or dimeric GEBP11 probes. Dimeric GEBP11 probe showed prolonged and stronger NIR signals than mono GEBP11 probe. Biodistribution assay suggested that GEBP11 NIR probes were enriched in gastric cancer xenografts. Using dimeric GEBP11 NIR probes in real-time surgery, the tumor margins and peritoneal metastases could be clearly visualized. Histological examination confirmed the complete resection of the tumor. (GEBP11)2-ACP-Cy5.5 could be a potential useful probe for intraoperative florescence guidance in gastric cancer surgery.

Targeting the insulin receptor with hormone and peptide dimers.

Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.

Neurotrophic Activity and Its Modulation by Zinc Ion of a Dimeric Peptide Mimicking the Brain-Derived Neurotrophic Factor N-Terminal Region.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin (NT) essential for neuronal development and synaptic plasticity. Dysregulation of BDNF signaling is implicated in different neurological disorders. The direct NT administration as therapeutics has revealed to be challenging. This has prompted the design of peptides mimicking different regions of the BDNF structure. Although loops 2 and 4 have been thoroughly investigated, less is known regarding the BDNF N-terminal region, which is involved in the selective recognition of the TrkB receptor. Herein, a dimeric form of the linear peptide encompassing the 1-12 residues of the BDNF N-terminal (d-bdnf) was synthesized. It demonstrated to act as an agonist promoting specific phosphorylation of TrkB and downstream ERK and AKT effectors. The ability to promote TrkB dimerization was investigated by advanced fluorescence microscopy and molecular dynamics (MD) simulations, finding activation modes shared with BDNF. Furthermore, d-bdnf was able to sustain neurite outgrowth and increase the expression of differentiation (NEFM, LAMC1) and polarization markers (MAP2, MAPT) demonstrating its neurotrophic activity. As TrkB activity is affected by zinc ions in the synaptic cleft, we first verified the ability of d-bdnf to coordinate zinc and then the effect of such complexation on its activity. The d-bdnf neurotrophic activity was reduced by zinc complexation, demonstrating the role of the latter in tuning the activity of the new peptido-mimetic. Taken together our data uncover the neurotrophic properties of a novel BDNF mimetic peptide and pave the way for future studies to understand the pharmacological basis of d-bdnf action and develop novel BDNF-based therapeutic strategies.

Design, Synthesis and DNA-Binding Activities of GCN4 Peptide Dimer Studied by Circular Dichroism Spectroscopy

Abstract: Basic region-leucine zipper (bZIP) family of DNA-binding protein GCN4 truncate Ac-[L250) GCN4 (226--252)-GGC-OH and its two designed analogues were synthesized by solid phase method using Fmoc­ strategy. Circular dichroism spectroscopy measurements showed that the DNA-binding affinity with target AP- 1 site was reduced when Thr-236 in GCN4 basic region was replaced by valine or glutamine.

A synthetic bivalent peptide ligand of EphB4 with potent agonistic activity

Interaction between ephrin receptor EphB4 and its ligand EFNB2 mediates bidirectional signaling important for cancer: forward EFNB2-to-EphB4 signaling that is tumor suppressive, and reverse EphB4-to-EFNB2 signaling that promotes angiogenesis important for tumor growth and metastasis. Molecular agents targeting these forward and reverse signals of EphB4-EFNB2 interaction can be used to probe the molecular mechanisms of these complex signaling pathways and develop new anticancer therapeutics. In this study, we applied a bivalent ligand design strategy to synthesize a novel dimeric peptide based on an antagonist TNYL-RAW. The dimeric peptide possessed higher EphB4 receptor binding affinity than the monomeric TNYL-RAW peptide. Interestingly, the dimerization of TNYL-RAW peptide converted a monomeric antagonist of EphB4 to a dimeric agonist. This dimeric agonist promoted EphB4 phosphorylation, internalization and degradation, reduced cancer cell motility, and inhibited tube formation of HUVEC. To investigate the mechanism of action of this bivalent dimeric peptide, FRET experiments and molecular dynamic simulation were conducted and suggested that this bivalent ligand recognizes two EphB4 simultaneously which may promote receptor dimerization and oligomerization. This was further supported by the study of this bivalent ligand containing deletion of critical residues on one of its monomers which impaired its simultaneous binding to two EphB4 and ability to cause EphB4 dimerization and phosphorylation. These results demonstrate the value of this novel bivalent agonist ligand of EphB4 as a probe of the bidirectional signaling of EphB4-EFNB2 and lead for cancer drug development.

The complex of miRNA2861 and cell-penetrating, dimeric α-helical peptide accelerates the osteogenesis of mesenchymal stem cells

BackgroundThe restoration of the functional ability of mesenchymal stem cells (MSCs) using epigenetic modification is very promising for patients with weak osteogenesis ability. This study focused on the acceleration of osteogenesis from MSCs using microRNA (miRNA)2861 and a cell-penetrating peptide (CPP), LK.MethodsWe performed MSCs penetration test of complex between the LK peptides and miRNA 2861. Three different experiments were performed to investigate the effects of miRNA 2861 on osteogenic differentiation in MSCs: 1) intensity of alizarin red staining, which reflects the status of mineralization by osteoblasts; 2) gene expression related to osteoblast differentiation; and 3) confirmation of corresponding protein translation for comparison with RNA expression levels.ResultsWe found that cLK effectively delivered miRNA 2861 into the cytoplasm of human MSCs and accelerated osteogenic differentiation from MSCs, as well as mineralization.ConclusionThe complex of miRNA 2861 with LK may have a positive effect on the osteogenic differentiation from MSCs and mineralization. Therapies using miRNAs combined with LK may be good candidates for the augmentation of osteogenesis in patients.

Concentration-Dependent Effects of Cholesterol on the Dimerization of Amyloid-β Peptides in Lipid Bilayers.

Membrane disruption mediated by the accumulation of amyloid-β (Aβ) on cell membranes is central to the pathogenesis of Alzheimer's disease (AD). Cholesterol, an important component of membranes, is well-recognized as a risk factor in AD. It can affect the aggregation and pore formation of Aβ on membranes whereas the specific effects are rather complex, particularly regarding the non-linear response to cholesterol concentrations. Yet, the mechanistic understanding of the role of cholesterol in Aβ-membrane interactions remains incomplete. Herein, we employed microsecond-scale molecular dynamics simulations to investigate the effects of cholesterol on Aβ dimerization in a lipid bilayer containing different molar ratios of cholesterol (0, 20, and 40 mol %). Cholesterol reduces the time required for the formation of stable dimers and exerts dual effects on Aβ-membrane interactions. First, cholesterol promotes the extraction of the C-terminal region from the membrane to water. Consequently, at the ratios of 0 and 20 mol %, peptides are anchored at the membrane-water interface, but they are repelled to water at a ratio of 40 mol % with high structural flexibility. Second, cholesterol weakens Aβ-membrane interactions, thereby enhancing inter-peptide interactions. The former is favorable for dimerization while the latter is not. The balance between two factors eventually leads to a non-monotonic effect on the degree of dimerization, whereby the number of inter-peptide contacts is the largest at a cholesterol ratio of 20 mol %. These results provide atomistic insights into the regulation mechanism of Aβ42 aggregation by cholesterol and help to understand the pathological link between cholesterol and AD.

LfcinB-Derived Peptides: Specific and punctual change of an amino acid in monomeric and dimeric sequences increase selective cytotoxicity in colon cancer cell lines

We observed how a change of specific residues in LfcinB dimeric and palindromic sequences caused a notable increase in the cytotoxic activity against CaCo-2 cells while maintaining or even diminishing the level of the cytotoxic effect against normal fibroblast and HEK-293 cells. In both cases, the IC 50 of the peptides was reduced by more than half of the concentration, diminishing the IC 50 value from 150 µg/mL (101 µM) (LfcinB (21–25) Pal : RWQWRWQWR) to 60 µg/mL (42.8 µM) for the modified palindromic peptide 5 [A] LfcinB (21–25) Pal : RWQWAWQWR and the IC 50 from 125 µg/mL (LfcinB (20–30) 2 : (RRWQWRMKKLG) 2 -K-Ahx to 58 µg/mL (18 µM) for the modified dimeric peptide 26 [F] LfcinB (20–30) 2 : (RRWQWRFKKLG) 2 -K-Ahx. The cytotoxic effect of 26 [F] LfcinB (20–30) 2 and LfcinB (21–25) Pal peptides against colon cancer cell line HCT-116 was greater than the cytotoxic effect of these peptides against Caco-2 cells, suggesting that the cytotoxic effect of these peptides is selective for colon cancer cell lines. The cytotoxic effect of the peptides rapidly caused severe damage to the morphology of CaCo-2 cells, while the morphology of the normal fibroblast and HEK-293 cells was not affected. The dimeric modified peptide 26 [F] LfcinB (20–30) 2 mainly caused death through apoptotic events. As for the palindromic modified peptide 5 [A] LfcinB (21–25) Pal , the cell death was induced by both necrotic and apoptotic pathways. All this suggests that specific modification of a single residue in the peptide sequence can improve the anticancer activity of the original monomeric or dimeric peptides, giving place to new potential molecules for the future development of drugs for use against colorectal carcinoma. Our results show that changes to a residue of the anti-cancer peptide sequence may be considered a versatile, feasible, and invaluable strategy for obtaining promising sequences for developing peptide-based cancer treatments.

(Keynote) An in Situ View of Nucleation and Coarsening at Solid-Electrolyte Interfaces

Nucleation of crystalline films at liquid-solid interfaces is widespread in both natural and synthetic systems. Observing this process is challenging, because it is a consequence of unstable density fluctuations, making the structures and events that must be probed both transient in nature and small in extent. Thus, the application of high-speed, molecularly resolved AFM to systems that assemble on surfaces provides a powerful method to quantify the dynamics and extract the underlying physics. Here we illustrate the fundamental insights into the pathways and energetics of nucleation and self-assembly made possible by this method using results from two contrasting systems: gibbsite (Al(OH)3) films grown on muscovite mica (001) and 2D arrays of peptides that self-assemble on both MoS2 (0001) and graphite. The results on gibbsite show that a dynamic population of clusters is created by continuous attachment and detachment of adsorbed ions, consistent with the basic concept of classical nucleation theory (CNT). However, severe discrepancies emerge when the values of key thermodynamic parameters are extracted from the data. Comparison to kinetic Monte Carlo simulations shows that electrostatic interactions between positively charged aluminum hydroxide clusters and the negative surface potential of mica is the source of these discrepancies, modifying both the chemical potential of the accumulating ions and the edge tension of the clusters. These interactions stabilize a nanostructured surface mesophase, which evolves gradually via a process of cluster growth and coalescence not limited by formation of a critically sized nucleus. The results for peptides on MoS2 show that the high anisotropy of inter-peptide interactions drives 2D assembly to occur row-by-row, starting with 1D nuclei. In agreement with expectations of CNT, these 1D nuclei form without a free energy barrier and there is no critical size. However, on graphite, two new phenomena emerge. First, peptide rows become highly mobile, enabling to a molecular level form of growth by oriented attachment. Second, appearance of the final phase is preceded by formation of a metastable phase, which dissolves as the stable phase nucleates. This manifestation of Ostwald’s Rule of Stages cannot be rationalized with the usual thermodynamic arguments due to the barrier-free nature of 1D nucleation. Instead, we propose a rationale rooted in chemical kinetics and applied here to peptide dimer formation at the surface.

A vitronectin-derived dimeric peptide suppresses osteoclastogenesis by binding to c-Fms and inhibiting M-CSF signaling

Vitronectin is an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bone, and is involved in diverse physiological processes. Here, we developed a new bioactive dimeric peptide (VnP-8-DN1 dimer) from a human vitronectin-derived motif (IDAAFTRINCQG; residues 206–217; VnP-8) via removal of an isoleucine residue at the N-terminus of VnP-8 and spontaneous air oxidation. The VnP-8-DN1 dimer potently enhanced cell attachment activity, and this activity was mediated by binding to cellular heparan sulfate proteoglycan receptors. Moreover, the VnP-8-DN1 dimer suppressed osteoclast differentiation by blocking the early stage of osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Furthermore, the VnP-8-DN1 dimer decreased the bone-resorbing activity of osteoclasts and increased the survival of osteoclast precursor cells by decreasing the cellular level of c-Fms and reducing RANK expression. Taken together, these results demonstrate that the VnP-8-DN1 dimer inhibits the early stages of M–CSF– and RANK-induced osteoclast differentiation by binding to c-Fms and inhibiting M-CSF signaling.

On-Resin Recognition of Aromatic Oligopeptides and Proteins through Host-Enhanced Heterodimerization

Peptide dimerization is ubiquitous in natural protein conjugates and artificial self-assemblies. A major challenge in artificial systems remains achieving quantitative peptide heterodimerization, critical for next-generation biomolecular purification and formulation of therapeutics. Here, we employ a synthetic host to simultaneously encapsulate an aromatic and a noncanonical l-perfluorophenylalanine-containing peptide through embedded polar−π interactions, constructing an unprecedented series of heteropeptide dimers. To demonstrate the utility, this heteropeptide dimerization strategy was applied toward on-resin recognition of N-terminal aromatic residues in peptides as well as insulin, both exhibiting high recycling efficiency (>95%). This research unveils a generic approach to exploit quantitative heteropeptide dimers for the design of supramolecular (bio)systems.

Amyloid-beta 16-22 peptide dimer simulation (150mM NaCl) with the CHARMM36m force field and Gromacs (Run 3)

Amyloid-beta 16-22 peptide dimer simulation (150mM NaCl) with the CHARMM36m force field and Gromacs (Run 3)

O-64 - A clickable C-glycosyl scaffold for RGD peptide dimer labelling and application in bimodal [18F]PET/NIRF imaging

O-64 - A clickable C-glycosyl scaffold for RGD peptide dimer labelling and application in bimodal [18F]PET/NIRF imaging

Correlation Study between Immunohistochemical Expression of Testicular Inhibin and Seminal Fluid Analysis

The inhibin family of dimeric peptide hormones are produced in the testis and are postulated to have paracrine and autocrine roles in the regulation of steroidogenesis, Testis is a reproductive organ composed of the seminiferous tubules which is the site of spermatogenesis, while the Sertoli and Leydig cells are responsible for secreting hormone; (inhibin), to maintain spermatogenesis. Dissimilarity of activin expression and plasma level it can cause variation to the parameters (sperm count, motility, germinal layer width). Aim of study was evaluation of the inhibin immunohistochemical activity in testicular tissue and the association between the inhibin expression and spermatogenesis. This study was performed in Al-Nahrain University / College of Medicine from November 2020 to June 2021. A total of 20 adult male rabbit. All animals were examined and monitored in order to exclude unhealthy animal. The animals were euthanized by inhalation of chloroform. The animals’ behavior observed during the experiment, in addition, we measured the weights of animals before and after the experiment. Dissection of the Rabbit was done to obtain the testis. We collected seminal fluid by milking the epididymis and then seminal fluid analysis was performed by professional lab technician. The volume of testis was measured. The specimens are kept in 10% formalin for (24-48) hours for histological preparation. In comparison between the two groups of the study (A, B) showed a statistically significant differences in means of sperm count, motility of sperm, germinal layer width, Leydig cell number, and Sertoli cell number between them. Regarding the means of inhibin expression and plasma level in the parameters (sperm count, motility of sperm, germinal layer width) were suggestively advanced in group B compared to that in group A. In conclusion, the expression and level of inhibin subunits, is correlated with parameters (sperm count, motility, germinal layer width), Inhibin peptide hormone acts as inhibitor for FSH-pituitary secretion that in turn promote spermatogenesis. The study shown a highly significant differences of inhibin manufacture and action through spermatogenesis is occupied in the fine regulator of the seminiferous epithelium of the cycle. Also, this study showed a negative correlation between the parameters with inhibin.