Exploring the Interactions between Plant Proanthocyanidins and Thiabendazole: Insights from Isothermal Titration Calorimetry.

Anthelmintic resistance in gastrointestinal nematodes produces substantial challenges to agriculture, and new strategies for nematode control in livestock animals are called for. Natural compounds, including tannins, with proven anthelmintic activity could be a functional option as structurally diverse complementary compounds to be used alongside commercial anthelmintics. However, the dual use of two anthelmintic components requires an understanding of the pharmacological effects of the combination, while information concerning the interactions between plant-based polyphenols and commercial anthelmintics is scarce. We studied the direct interactions of proanthocyanidins (PAs, syn. condensed tannins) and a commercial anthelmintic thiabendazole, as a model substance of benzimidazoles, by isothermal titration calorimetry (ITC). Our results show evidence of a direct interaction of an exothermic nature with observed enthalpy changes ranging from 0 to -30 kJ/mol. The strength of the interaction between PAs and thiabendazole is mediated by structural characteristics of the PAs with the strongest positive correlation originating from the presence of galloyl groups and the increased degree of polymerization.

Tannins Can Have Direct Interactions with Anthelmintics: Investigations by Isothermal Titration Calorimetry.

Plant tannins are known for their anthelmintic and antiparasitic activities and have been increasingly studied to battle the ever-growing problem of anthelmintic resistance. While tannins have been shown to exhibit these activities on their own, one approach would be to use them as complementary nutrients alongside commercial anthelmintics. So far, research on the interactions between tannins and anthelmintics is limited, and few studies have reported both synergistic and antagonistic effects depending on the type of tannin and the method used. These interactions could either strengthen or weaken the efficacy of commercial anthelmintics, especially if tannin-rich diets are combined with anthelmintics used as oral drenches. To study these interactions, a series of hydrolysable tannins (HTs) was selected, and their direct interactions with thiabendazole (TBZ) were evaluated by isothermal titration calorimetry (ITC), which allowed the detection of the exothermic interaction but also the roles and significances of different structural features of HTs in these interactions. Our results show that HTs can have a direct interaction with the benzimidazole anthelmintic TBZ and that the interaction is strengthened by increasing the number of free galloyl groups and the overall molecular flexibility of HTs.

Extracellular citrullination inhibits the function of matrix associated TGF-ß.

In inflammatory arthritis peptidyl arginine deiminase (PAD) enzymes can citrullinate arginine residues in extracellular matrix (ECM) proteins, such as collagens and fibronectin. This may lead to the generation of anti-citrullinated protein antibodies, important diagnostic markers in rheumatoid arthritis. In addition, the citrullination may directly affect protein function. Based on structural analysis, we found that most ECM-associated growth factors (GFs) have arginine residues in their receptor recognition sites. Thus, they are potential functional targets of extracellular citrullination. To examine this further, we focused on the citrullination of transforming growth factor-ßs (TGF-ß), well-known ECM-associated GFs. PAD-treatment of CHO-LTBP1 cell derived matrix, rich with TGF-ß, decreased the level of TGF-ß activity as detected by HaCaT and MLEC-PAI-1/Lu reporter cells. Additional experiments indicated that PAD-treatment inhibits the integrin-mediated TGF-ß activation since PAD-treatment decreased the binding of integrin αVß6 ectodomain as well as integrin-mediated spreading of MG-63 and HaCaT cells to ß1-latency associated peptide (TGF-ß1 LAP). The citrullination of the RGD site, an important integrin recognition motif, was confirmed by mass spectrometry. Furthermore, the citrullination of active TGF-ß1 inhibited its binding to recombinant TGF-ß receptor II, and prevented its ability to activate TGF-ß signaling. Thus, extracellular PAD activity can affect the function of ECM-associated growth factors by different mechanisms. Importantly, the citrullination of both latent and active TGF-ß has the potency to regulate the inflammatory process.

Citrullination of collagen II affects integrin-mediated cell adhesion in a receptor-specific manner.

Citrullinated collagen II (CII) is a well-known autoantigen in rheumatoid arthritis (RA). However, the direct effects of CII citrullination on cell behavior have not been described. To study whether citrullination of CII could affect cellular functions, we measured the adhesion of 3 different cell types (human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal stem cells) with impedance-based technology. The binding of different collagen receptor integrins to citrullinated collagen was studied by CHO cell lines, each overexpressing 1 of the 4 human collagen receptors on the cell surface, and with solid-phase binding assays, using the recombinant human integrin α1I, α2I, α10I, and α11I domains. Collagen citrullination decreased the adhesion of synovial fibroblasts ∼50% (P<0.05) and mesenchymal stem cells ∼40% (P<0.05) by specifically decreasing the binding of integrins α10ß1 and α11ß1 to arginine-containing motifs, such as GFOGER. In contrast, citrullination had only a minor effect on the function of α1ß1 and α2ß1 integrins, which have been reported to play a critical role in regulating leukocyte function. Molecular modeling was used to explain the detected functional differences at the structural level. Given that the integrins regulate cell metabolism, proliferation, and migration, we suggest that collagen citrullination modifies the pathogenesis of RA. Here, CII citrullination was shown to decrease the survival of mesenchymal stem cells.

Molecular mechanism of T-cell protein tyrosine phosphatase (TCPTP) activation by mitoxantrone.

T-cell protein tyrosine phosphatase (TCPTP) is a ubiquitously expressed non-receptor protein tyrosine phosphatase. It is involved in the negative regulation of many cellular signaling pathways. Thus, activation of TCPTP could have important therapeutic applications in diseases such as cancer and inflammation. We have previously shown that the α-cytoplasmic tail of integrin α1ß1 directly binds and activates TCPTP. In addition, we have identified in a large-scale high-throughput screen six small molecules that activate TCPTP. These small molecule activators include mitoxantrone and spermidine. In this study, we have investigated the molecular mechanism behind agonist-induced TCPTP activation. By combining several molecular modeling and biochemical techniques, we demonstrate that α1-peptide and mitoxantrone activate TCPTP via direct binding to the catalytic domain, whereas spermidine does not interact with the catalytic domain of TCPTP in vitro. Furthermore, we have identified a hydrophobic groove surrounded by negatively charged residues on the surface of TCPTP as a putative binding site for the α1-peptide and mitoxantrone. Importantly, these data have allowed us to identify a new molecule that binds to TCPTP, but interestingly cannot activate its phosphatase activity. Accordingly, we describe here mechanism of TCPTP activation by mitoxantrone, the cytoplasmic tail of α1-integrin, and a mitoxantrone-like molecule at the atomic level. These data provide invaluable insight into the development of novel TCPTP activators, and may facilitate the rational discovery of small-molecule cancer therapeutics.

Leukocyte integrins αLß2, αMß2 and αXß2 as collagen receptors--receptor activation and recognition of GFOGER motif.

Integrins αLß2, αMß2 and αXß2 are expressed on leukocytes. Their primary ligands are counter transmembrane receptors or plasma proteins, such as intercellular cell adhesion molecule-1 (ICAM-1) or components of complement system (iC3b, iC4b), respectively. Function blocking antibodies for these integrins may also reduce cell adhesion to collagens. To make the first systematical comparison of human α(L)ß2, α(M)ß2 and α(X)ß2 as collagen receptors, we produced the corresponding integrin αI domains both in wild-type and activated form and measured their binding to collagens I-VI. In the "closed" (wild-type) conformation, the α(L)I and α(M)I domains bound with low avidity to their primary ligands, and the interaction with collagens was also very weak. Gain-of-function mutations α(L) I306G, α(L) K287C/K294C and α(M) I316G are considered to mimic "open", activated αI domains. The binding of these activated αI domains to the primary ligands was clearly stronger and they also recognized collagens with moderate avidity (K(d)400 nM). After activation, the αLI domain favored collagen I (K(d )≈ 80 nM) when compared to collagen IV. The integrin αXI domain acted in a very different manner since already in native, wild-type form it bound to collagen IV and iC3b (K(d) ≈ 200-400 nM). Antibodies against αXß2 and αMß2 blocked promyelocytic leukemia cell adhesion to the collagenous GFOGER motif, a binding site for the ß1 integrin containing collagen receptors. In brief, leukocyte ß2 integrins may act as collagen receptors in a heterodimer specific manner.

Fluorescent small molecule probe to modulate and explore α2ß1 integrin function.

Collagen binding integrins are an important family of cell surface receptors that mediate bidirectionally signals between the interior of the cell and the extracellular matrix. The protein-protein interactions between cells and collagen are necessary for many physiological functions, but also promote diseases. For example, the interaction of α2ß1 integrin and collagen has been shown to have an important role in thrombus formation and cancer spread. The fact that the discovery of small molecules that can block such protein-protein interactions is highly challenging has significantly hindered the discovery of pharmaceutical agents to treat these diseases. Here, we present a rationally designed novel fluorescent molecule that can be synthesized in just a few minutes from commercially available starting materials. This molecule blocks the protein-protein interaction between α2ß1 integrin and collagen, and due to its fluorescent properties, it can be employed in wide variety of biological applications.

Lumican inhibits cell migration through α2ß1 integrin.

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2ß1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2ß1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K(d)≥200nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2ß1 integrin.

Cellular receptors of extracellular matrix molecules.

Extracellular matrix (ECM) is composed of large collagen fibrils. Glycoproteins, such as fibronectin, can bind to collagen or form their own networks. Collagen fibrils are also decorated by proteoglycans, proteins that have large glycosaminoglycan sidechains. In addition, extracellular space often contains hyaluronan, a large glycosaminoglycan molecule that has no core protein. Basement membranes represent a specialized form of extracellular matrix. Basement membranes are built by laminin and type IV collagen networks. In multicellular animals cells are anchored to ECM and basement membranes. Cell locomotion during development and after tissue injury is also based on cellular interactions with different matrix molecules. Specific cell surface receptors mediate these interactions. The largest family of receptors, which mediates cell adhesion to fibronectin, laminins and collagens is called the integrins. Several other cellular receptors have also evolved to bind to various matrix components. Here, we review the basic facts about these receptors and shortly describe their role in human diseases, including cancer and inflammation.

The fibril-associated collagen IX provides a novel mechanism for cell adhesion to cartilaginous matrix.

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.

alpha 11beta 1 integrin recognizes the GFOGER sequence in interstitial collagens.

The integrins alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1) are referred to as a collagen receptor subgroup of the integrin family. Recently, both alpha(1)beta(1) and alpha(2)beta(1) integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, alpha(11)beta(1), recognizes collagens using C2C12 cells transfected with alpha(11) cDNA and the bacterially expressed recombinant alpha(11) I domain. The ligand binding properties of alpha(11)beta(1) were compared with those of alpha(2)beta(1). Mg(2+)-dependent alpha(11)beta(1) binding to type I collagen required micromolar Ca(2+) but was inhibited by 1 mm Ca(2+), whereas alpha(2)beta(1)-mediated binding was refractory to millimolar concentrations of Ca(2+). The bacterially expressed recombinant alpha(11) I domain preference for fibrillar collagens over collagens IV and VI was the same as the alpha(2) I domain. Despite the difference in Ca(2+) sensitivity, alpha(11)beta(1)-expressing cells and the alpha(11) I domain bound to helical GFOGER sequences in a manner similar to alpha(2)beta(1)-expressing cells and the alpha(2) I domain. Modeling of the alpha I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by alpha(1)beta(1), alpha(2)beta(1), and also alpha(11)beta(1) integrins. Although alpha(10) and alpha(11) chains show the highest sequence identity, alpha(2) and alpha(11) are more similar with regard to collagen specificity. Future studies will reveal whether alpha(2)beta(1) and alpha(11)beta(1) integrins also show overlapping biological functions.