Heparin-binding Domain Research Articles

Pharmacological Impacts of Mucopolysacccharide Polyphosphates in the Epidermis Involves Inhibition of Amphiregulin-Mediated Signals in Keratinocytes.

The epidermis, the most superficial layer of the human skin, serves a critical barrier function, protecting the body from external pathogens and allergens. Dysregulation of epidermal differentiation contributes to barrier dysfunction and has been implicated in the pathology of various dermatological diseases, including atopic dermatitis (AD). Mucopolysaccharide polysulphate (MPS) is a moisturising agent used to treat xerosis in patients with AD. However, its mechanism of action on keratinocytes, the main constituents of the epidermis, remains unclear. In this study, we investigated the effect of MPS on keratinocytes by subjecting adult human epidermal and three-dimensional cultured keratinocytes to MPS treatment, followed by transcriptome analysis. The analysis revealed that MPS treatment enhances keratinocyte differentiation and suppresses proliferation. We focused on amphiregulin (AREG), a membrane protein that belongs to the epidermal growth factor (EGF) family and possesses a heparin-binding domain, as a significant target among the genes altered by MPS. MPS exerted an inhibitory effect directly on AREG, rather than on EGF receptors or other members of the EGF family. Furthermore, AREG leads to a reduction in epidermal barrier function, whereas MPS contributes to barrier enhancement via AREG inhibition. Collectively, these findings suggest that MPS modulates barrier function through AREG inhibition, offering insights into potential therapeutic strategies for skin barrier restoration.

Ultrasound-Responsive HBD Peptide Hydrogel with Antibiofilm Capability for Fast Diabetic Wound Healing.

Despite advancements in therapeutic agents for diabetic chronic wounds, challenges such as suboptimal bioavailability, intricate disease milieus, and inadequate delivery efficacy have impeded treatment outcomes. Here, ultrasound-responsive hydrogel incorporated with heparin-binding domain (HBD) peptide nanoparticles is developed to promote diabetic wound healing. HBD peptide, derived from von Willebrand Factor with angiogenic activity, are first engineered to self-assemble into nanoparticles with enhanced biostability and bioavailability. Ultrasound responsive cargo release and hydrogel collapses are first verified through breakage of crosslinking. In addition, desired antioxidant and antibacterial activity of such hydrogel is observed. Moreover, the degradation of hydrogel under ultrasound stimulation into smaller fragments facilitated the deeper wound penetration of ≈400 µm depth. Complete wound closure is observed from diabetic mice with chronic wounds after being treated with the proposed hydrogel. In detail, in vivo studies revealed that hydrogels loaded with HBD peptide nanoparticles increased the levels of angiogenesis-related growth factors (VEGF-A, CD31, and α-SMA) to effectively accelerate wound repair. Overall, this study demonstrates that ultrasound-responsive HBD peptide hydrogel provides a synergistic therapeutic strategy for external biofilm elimination and internal effective delivery for diabetic wounds with biofilm infection.

Identification of small molecule antagonists of sonic hedgehog/heparin binding with activity in hedgehog functional assays

Sonic hedgehog (Shh) is a morphogen with important roles in embryonic development and in the development of a number of cancers. Its activity is modulated by interactions with binding partners and co-receptors including heparin and heparin sulfate proteoglycans (HSPG). To identify antagonists of Shh/heparin binding, a diverse collection of 34,560 chemicals was screened in single point 384-well format. We identified and confirmed twenty six novel small molecule antagonists with diverse structures including four scaffolds that gave rise to multiple hits. Nineteen of the confirmed hits blocked binding of the N-terminal fragment of Shh (ShhN) to heparin with IC50 values < 50 μM. In the Shh-responsive C3H10T1/2 cell model, four of the compounds demonstrated the ability to block ShhN-induced alkaline phosphatase activity. To demonstrate a direct and selective effect on ShhN ligand mediated activity, two of the compounds were able to block induction of Gli1 mRNA, a primary downstream marker for Shh signaling activity, in Shh-mediated but not Smoothened agonist (SAG)-mediated C3H10T1/2 cells. Direct binding of the two compounds to ShhN was confirmed by thermal shift assay and molecular docking simulations, with both compounds docking with the N-terminal heparin binding domain of Shh. Overall, our findings indicate that small molecule compounds that block ShhN binding to heparin and act to inhibit Shh mediated activity in vitro can be identified. We propose that the interaction between Shh and HSPGs provides a novel target for identifying small molecules that bind Shh, potentially leading to novel tool compounds to probe Shh ligand function.

Unlocking precision gene therapy: harnessing AAV tropism with nanobody swapping at capsid hotspots.

Adeno-associated virus (AAV) has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2/5-fold wall and 5-fold pore of the AAV capsid that are amenable to insertion of larger protein domains, including nanobodies. Here, we demonstrate that these hotspots facilitate AAV tropism switching through simple nanobody replacement without extensive optimization in both VP1 and VP2. Our data suggest that engineering VP2 is the preferred path for maintaining both virus production yield and infectivity. We demonstrate highly specific targeting of human cancer cells expressing fibroblast activating protein (FAP). Furthermore, we found that the combination of FAP nanobody insertion plus ablation of the heparin binding domain can reduce off-target infection to a minimum, while maintaining a strong infection of FAP receptor-positive cells. Taken together, our study shows that nanobody swapping at multiple capsid locations is a viable strategy for nanobody-directed cell-specific AAV targeting.

The heparin-binding domain of VEGF165 directly binds to integrin αvβ3 and VEGFR2/KDR D1: a potential mechanism of negative regulation of VEGF165 signaling by αvβ3.

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1-110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111-165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

The Evolution and Application of a Novel DNA Aptamer Targeting Bone Morphogenetic Protein 2 for Bone Regeneration.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

Exploring single-molecule interactions: heparin and FGF-1 proteins through solid-state nanopores.

Detection and characterization of protein-protein interactions are essential for many cellular processes, such as cell growth, tissue repair, drug delivery, and other physiological functions. In our research, we have utilized emerging solid-state nanopore sensing technology, which is highly sensitive to better understand heparin and fibroblast growth factor 1 (FGF-1) protein interactions at a single-molecule level without any modifications. Understanding the structure and behavior of heparin-FGF-1 complexes at the single-molecule level is very important. An abnormality in their formation can lead to life-threatening conditions like tumor growth, fibrosis, and neurological disorders. Using a controlled dielectric breakdown pore fabrication approach, we have characterized individual heparin and FGF-1 (one of the 22 known FGFs in humans) proteins through the fabrication of 17 ± 1 nm nanopores. Compared to heparin, the positively charged heparin-binding domains of some FGF-1 proteins translocationally react with the pore walls, giving rise to a distinguishable second peak with higher current blockade. Additionally, we have confirmed that the dynamic FGF-1 is stabilized upon binding with heparin-FGF-1 at the single-molecule level. The larger current blockades from the complexes relative to individual heparin and the FGF-1 recorded during the translocation ensure the binding of heparin-FGF-1 proteins, forming binding complexes with higher excluded volumes. Taken together, we demonstrate that solid-state nanopores can be employed to investigate the properties of individual proteins and their complex interactions, potentially paving the way for innovative medical therapies and advancements.

Ultrahigh-Efficacy VEGF Neutralization Using Carbonized Nanodonuts: Implications for Intraocular Anti-Angiogenic Therapy.

Ocular angiogenesis, associated with diseases such as retinopathy of prematurity and diabetic retinopathy, is a leading cause of irreversible vision loss. Herein, carbon nanodonuts (CNDs) with a donut-shaped structure are synthesized using sodium alginate (SA) and 1,8-diaminooctane (DAO) through a one-step thermal process. The formation of SA/DAO-CNDs occurs through a crosslinking reaction between SA and DAO, creating amide bonds followed by partial carbonization. In human retinal pigment epithelialcells exposed to H2 O2 or lipopolysaccharide, the SA/DAO-CNDs display a more than fivefold reduction in reactive oxygen species and proinflammatory cytokines, such as IL-6 and IL-1β, when compared to carbonized nanomaterials produced exclusively from SA. Furthermore, the CNDs effectively inhibit vascular endothelial growth factor A-165 (VEGF-A165 )-induced cell migration and tube formation in human umbilical vein endothelial cells due to their strong affinity for VEGF-A165 , with a dissociation constant of 2.2 × 10-14 M, over 1600 times stronger than the commercial drug bevacizumab (Avastin). Trypsin digestion coupled with LC-MS/MS analysis reveals that VEGF-A165 interacts with SA/DAO-CNDs through its heparin-binding domain, leading to activity loss. The SA/DAO-CNDs demonstrate excellent biocompatibility and potent anti-angiogenic effects in chicken embryos and rabbit eyes. These findings suggest that SA/DAO-CNDs hold promise as a therapeutic agent for treating various angiogenesis-related ocular diseases.

Design of an Anti-HMGB1 Synthetic Antibody for In Vivo Ischemic/Reperfusion Injury Therapy.

High-mobility group box 1 (HMGB1) is a multifunctional protein. Upon injury or infection, HMGB1 is passively released from necrotic and activated dendritic cells and macrophages, where it functions as a cytokine, acting as a ligand for RAGE, a major receptor of innate immunity stimulating inflammation responses including the pathogenesis of cerebral ischemia/reperfusion (I/R) injury. Blocking the HMGB1/RAGE axis offers a therapeutic approach to treating these inflammatory conditions. Here, we describe a synthetic antibody (SA), a copolymer nanoparticle (NP) that binds HMGB1. A lightly cross-linked N-isopropylacrylamide (NIPAm) hydrogel copolymer with nanomolar affinity for HMGB1 was selected from a small library containing trisulfated 3,4,6S-GlcNAc and hydrophobic N-tert-butylacrylamide (TBAm) monomers. Competition binding experiments with heparin established that the dominant interaction between SA and HMGB1 occurs at the heparin-binding domain. In vitro studies established that anti-HMGB1-SA inhibits HMGB1-dependent ICAM-1 expression and ERK phosphorylation of HUVECs, confirming that SA binding to HMGB1 inhibits the proteins' interaction with the RAGE receptor. Using temporary middle cerebral artery occlusion (t-MCAO) model rats, anti-HMGB1-SA was found to accumulate in the ischemic brain by crossing the blood-brain barrier. Significantly, administration of anti-HMGB1-SA to t-MCAO rats dramatically reduced brain damage caused by cerebral ischemia/reperfusion. These results establish that a statistical copolymer, selected from a small library of candidates synthesized using an "informed" selection of functional monomers, can yield a functional synthetic antibody. The knowledge gained from these experiments can facilitate the discovery, design, and development of a new category of drug.

Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection

ABSTRACTDisadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

Heparin‐guided binding of vascular endothelial growth factor to supramolecular biomaterial surfaces

AbstractGrowth factors can steer the biological response to a biomaterial post implantation. Heparin is a growth factor binding molecule that can coordinate growth factor presentation to cells and therefore is able to regulate many biological processes. One way to functionalize biomaterials with heparin and growth factors is via a supramolecular approach. Here, we show a proof‐of‐concept study in which a supramolecular approach based on ureido‐pyrimidinone (UPy) was used, which allows for modular functionalization. PCLdiUPy was functionalized with a UPy‐modified heparin binding peptide (UPy‐HBP) to facilitates binding of heparin, which in turn can bind vascular endothelial growth factor (VEGF) via its heparin binding domain. The adsorption of both heparin and VEGF were studied in two different functionalization approaches (pre‐complex and two‐step) and at different molecular ratios. Quartz crystal microbalance with dissipation energy adsorption data showed that VEGF and pre‐complexed heparin:VEGF adsorbed non‐specifically, with no distinguish between non‐specific adsorption and heparin guided‐adsorption. On the biological side, heparin guided‐adsorption of Heparin:VEGF enhanced HUVECs surface coverage as compared to non‐specific adsorption. These results provide a detailed insight on the molecular sandwich which is useful for new design strategies of supramolecular biomaterials with well‐controlled immobilization of different growth factors.

Adsorption of Heparin-Binding Fragments of Fibronectin onto Hydrophobic Surfaces

Fibronectin is a multi-domain, extracellular matrix protein that plays a number of biological roles. As the adsorption of fibronectin onto the surface of implanted devices can lead to an inflammatory response or bacterial colonisation, understanding the interaction of fibronectin with material surfaces is important in the design of materials for biomedical applications. This, however, relies on having knowledge of the molecular-scale behaviour of proteins, which is difficult to investigate experimentally. In this paper, we used molecular dynamics simulations to investigate the adsorption of heparin-binding fibronectin domains onto hydrophobic surfaces. Despite the high similarity between these, their adsorption differs both in terms of the strength and the specificity of this, indicating that relatively small changes in protein structure can lead to significant changes in adsorption behaviour. This suggests that the interplay between protein structure and surface chemistry is vital for understanding the protein adsorption process and the design of novel biomaterials.

Mechanical Signal-Tailored Hydrogel Microspheres Recruit and Train Stem Cells for Precise Differentiation.

The aberrant mechanical microenvironment in degenerated tissues induces misdirection of cell fate, making it challenging to achieve efficient endogenous regeneration. Herein, a hydrogel microsphere-based synthetic niche with integrated cell recruitment and targeted cell differentiation properties via mechanotransduction was constructed for enhanced endogenous regeneration. Through the incorporation of microfluidics and photo-polymerization strategies, we prepared fibronectin (Fn) modified methacrylated gelatin (GelMA) microspheres with the independently tunable elastic modulus(1-10Kpa) and ECM ligand density (2 and 10μg/ml), allowing a wide range of cytoskeleton modulation to trigger the corresponding mechanobiological signaling to direct cell fate. The combination of the soft matrix (2Kpa) and low ligand density (2μg/ml) could support the nucleus pulposus (NP)-like differentiation of intervertebral disc (IVD) progenitor/stem cells by translocating Yes-associated protein (YAP), without the addition of inducible biochemical factors. Meanwhile, platelet-derived growth factor-BB was loaded onto Fn-GelMA microspheres (PDGF@Fn-GelMA) via the heparin-binding domain of Fn and exhibited continuous release for over 28 days to initiate endogenous cell recruitment. In in vivo experiments, hydrogel microsphere-niche maintained the IVD structure and stimulated ECM synthesis, thereby enhancing the endogenous regeneration of NP. Overall, this synthetic niche with cell recruiting and mechanical training capabilities offered a promising novel strategy for endogenous tissue regeneration. This article is protected by copyright. All rights reserved.

C-Terminal Fibronectin Exerts Beneficial Effects in Reducing Tissue Damage and Modulating Macrophage Function in a Murine Septic Model.

Fibronectin (FN) can improve organ function and slow the progression of sepsis, but full-length FN is hard to be exacted as a therapeutic. This study aimed to investigate the beneficial effects of C-terminal heparin-binding domain polypeptide of FN (rhFNHC-36) in a cecal ligation and puncture (CLP)-mediated murine septic model and explore its regulatory effects on macrophages. Mice were randomly assigned to four groups: unoperated control (Normal), sham operation control (Sham), CLP-operation with intravenous injection of phosphate-buffered saline (CLP+PBS), and CLP-operation with rhFNHC-36 treatment (CLP+rhFNHC-36). Blood and abdominal fluid samples were subjected to bacterial colony formation assays. Organs (liver, spleen, and lung) were undergone histopathological analyses and/or weighed to obtain organ indices. Serum interleukin-6 (IL-6) levels, nitric oxide (NO) release from isolated abdominal macrophages, and chemotactic effect of macrophages were measured with commercial kits. Surface programmed death ligand 1 (PD-L1) expression on macrophages was measured by flow cytometry. Mice in the CLP+PBS group showed a lower survival rate than that in the CLP+rhFNHC-36 group. Improved survival was associated with better clearance of bacterial pathogens, as evidenced by colony formation assays. The CLP-induced decrease in thymus and spleen indices was attenuated by rhFNHC-36 treatments. rhFNHC-36 alleviated sepsis-associated tissue damage in liver, spleen, and lung. CLP-mediated increases in plasma IL-6 levels were reversed by rhFNHC-36 treatment. NO levels in peritoneal macrophages after lipopolysaccharides (LPS)-stimulation in the CLP+rhFNHC-36 group were lower than that in the CLP+PBS group. Notably, macrophages from the CLP+rhFNHC-36 group retained better chemotaxis ability. After LPS challenge, these macrophages had a reduced percentage of PD-L1-positive cells compared to those in the CLP+PBS group. rhFNHC-36 improved survival of mice with CLP-induced sepsis by reducing tissue damage and modulating macrophage function. Our work provides critical insight for developing FN-based and macrophages-targeted therapeutics for treating sepsis.

Cellular infiltration in an injectable sulfated cellulose nanocrystal hydrogel and efficient angiogenesis by VEGF loading

BackgroundCellular infiltration and angiogenesis into implanted biomaterial scaffolds are crucial for successful host tissue integration and tissue regeneration. Cellulose nanocrystal (CNC) is a nano-sized cellulose derivative, which can form an injectable physical gel with salts. Sulfate groups of sulfated CNC (CNC-S) can act as a binding domain to various growth factors and cytokines with a heparin-binding domain for sustained release of them. Vascular endothelial growth factor (VEGF) can promote the proliferation of endothelial cells and angiogenesis. In this study, VEGF-loaded CNC-S hydrogel was evaluated as an injectable scaffold that can induce cellular infiltration and angiogenesis.MethodsCNC-S was hydrolyzed to get desulfated CNC (CNC-DS), which was used as a negative control group against CNC-S. Both CNC-S and CNC-DS hydrogels were prepared and compared in terms of biocompatibility and VEGF release. The hydrogels with or without VEGF loading were subcutaneously injected into mice to evaluate the biocompatibility, cellular infiltration, and angiogenesis induction of the hydrogels.ResultsBoth hydrogels possessed similar stability and shear-thinning behavior to be applicable as injectable hydrogels. However, CNC-S hydrogel showed sustained release (until 8 weeks) of VEGF whereas CNC-DS showed a very fast release of VEGF with a large burst. Subcutaneously injected CNC-S hydrogel showed much enhanced cellular infiltration as well as better biocompatibility with milder foreign body response than CNC-DS hydrogel. Furthermore, VEGF-loaded CNC-S hydrogel induced significant angiogenesis inside the hydrogel whereas VEGF-loaded CNC-DS did not.ConclusionCNC-S possesses good properties as a biomaterial including injectability, biocompatibility, and allowing cellular infiltration and sustained release of growth factors. VEGF-loaded CNC-S hydrogel exhibited efficient angiogenesis inside the hydrogel. The sulfate group of CNC-S was a key for good biocompatibility and the biological activities of VEGF-loaded CNC hydrogel.

Molecular mechanism of binding between a therapeutic RNA aptamer and its protein target VEGF: A molecular dynamics study.

Macugen is a therapeutic RNA aptamer against vascular endothelial growth factor (VEGF)-165, the VEGF isoform primarily responsible for angiogenesis. It has been reported that Macugen inhibits angiogenesis by specifically binding to the heparin binding domain (HBD) of VEGF165. The mechanism of the molecular recognition between HBD and Macugen is investigated here using all-atom molecular dynamics simulations. We find that Macugen recognizes HBD by an induced-fit mechanism with major conformational changes in Macugen and almost no changes in the structure of HBD, whereas HBD recognizes Macugen by a conformational selection mechanism.

The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

Filamentous Hemagglutinin of Bordetella pertussis Does Not Interact with the β2 Integrin CD11b/CD18.

The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the β2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the β2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.

Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called “NT-mini” and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT’s heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

The Relationship between Mutations in Gene-Specific Domains of Salivary Fibronectin (cFn) and Dynamin-2 (Dynm-2) and the Development of Porphyromonas gingivalis-Initiated Periodontitis

Periodontitis is a chronic inflammatory disease characterized by the destruction of the supporting structures of the teeth. Its high prevalence and negative effects on quality of life make it one of the current problems in dentistry. Porphyromonas gingivalis (P. gingivalis) is the predominant periodontal pathogen that expresses a number of virulence factors involved in the pathogenesis of periodontitis. P. gingivalis fimbriae are a critical factor in the interaction between the organism and the host tissue. They promote both bacterial adhesion and invasion into the target sites. Fimbriae are capable of binding to human saliva components, extracellular matrix proteins, and commensal bacteria, as well as firmly binding to the cellular integrin α5β1. After attachment to α5β1-integrin, P. gingivalis is captured by cellular pseudopodia, which makes invagination through an actin-mediated pathway possible. It has been proven that the invagination event also requires the participation of the host cell dynamin, actin fibers, microtubules and lipid rafts. Work has emerged investigating mutations in the proline-rich terminal domain (PRD) and their impact on disease development. Salivary antimicrobial peptides are early protective factors against microbial attack. Of great interest is fibronectin (FN) as the main competitor of P. gingivalis fimbriae. The FN can interact with cells in three different regions: the central cell-binding domain (CCBD), the COOH terminal heparin-binding domain (Hep2), and the type III connecting segment (IIICS), including the CS1 region (Yamada, 1991). CCBD is the major cell-adhesion domain of FN and contains an Arg–Gly–Asp (RGD) motif that is recognized by members of the cell adhesion receptor integrin family, including a5b1, which is the primary FN receptor in many cell types. The work focuses on identifying the relationship between the development of periodontitis and the presence of mutations in the adhesion domains of salivary proteins such as cellular fibronectin (cFN) and dynamin-2 (DYNM2).