In silico study suggests potential drugs that target CD151 to treat breast cancer and glioblastoma.

Recently tetraspanin CD151 has been identified as an important biological target involved in metastatic processes which include cell adhesion, tumor progression processes, and so forth in different types of cancers, such as breast cancer and glioblastoma. This in Silico study considered 1603 compounds from the Food and Drug Administration database, after performing an ADMET analysis; we selected 853 ligands, which were used for docking analysis. The most promising ligands were selected from docking studies, based on two criteria: (a) showed lowest affinity to the CD151 protein and (b) they interact with the QRD motif, located in the second extracellular loop. Furthermore, we investigate the stability of the protein-ligand complexes through MD simulations as well as free energy MM-PBSA calculations. From these results, loperamide and glipizide were identified as the best evaluated drugs. We suggest an in vitro analysis is needed to confirm our in silico prediction studies.

Expression and distribution of CD151 as a partner of alpha6 integrin in male germ cells.

The physiological importance of CD151 tetraspanin is known from somatic cells and its outside-in signalling through integrins was described. In male germ cells, two tetraspanins, CD9 and CD81, are involved in sperm-egg membrane fusion, and similarly to integrins, they occupy characteristic regions. We report here on a newly discovered presence of CD151 in sperm, and present its expression and distribution during spermatogenesis and sperm transition during the acrosome reaction. We traced CD151 gene and protein expression in testicular cell subpopulations, with strong enrichment in spermatogonia and spermatids. The testicular and epididymal localization pattern is designated to the sperm head primary fusion site called the equatorial segment and when compared to the acrosome vesicle status, CD151 was located into the inner acrosomal membrane overlying the nucleus. Moreover, we show CD151 interaction with α6 integrin subunit, which forms a dimer with ß4 as a part of cis-protein interactions within sperm prior to gamete fusion. We used mammalian species with distinct sperm morphology and sperm maturation such as mouse and bull and compared the results with human. In conclusion, the delivered findings characterise CD151 as a novel sperm tetraspanin network member and provide knowledge on its physiology in male germ cells.

Peptides of tetraspanin oncoprotein CD151 trigger active immunity against primary tumour and experimental lung metastasis.

BACKGROUND: Active immunotherapy is an effective, long-lasting, cheap, and safe approach to suppress cancer progression; however, the key issue is to develop appropriate tumour vaccines. Oncoproteins are up-regulated under various stress conditions and promote cell survival. Oncoproteins and their immunogenic domains could serve well as tumour vaccines and prime the hosts' active anti-tumour immunity. METHODS: Proteomic and bioinformatic analyses were performed to identify potential tumour associated antigens (TAAs). Then, peptides derived from CD151 were designed and synthesized according to the major histocompatibility complex (MHC) I binding and immunogenicity. Cytotoxicity assay, flow cytometry, immunohistochemistry, and in vivo bioluminescence imaging were performed to assess the active anti-tumour immunity triggered by CD151 peptides in H22 primary hepatoma and experimental 4T1 breast cancer lung metastasis models. FINDINGS: CD151 was identified as an ideal TAA based on proteomic and bioinformatic analyses. CD151 peptides as tumour vaccines triggered active anti-tumour immunity against H22 hepatoma and the lung metastasis of 4T1 breast cancer in two mouse models through the activation of CD8+IFNγ+ lymphocytes and the subsequent targeted cytotoxicity. Further, the peptides suppressed the negative regulators, myeloid-derived suppressor cells. Survival was prolonged for mice with lung metastases from CD151 peptide-immunised groups. INTERPRETATION: The up-regulated oncoproteins in 8 Gy-irradiated tumour cells are good candidates for designing immunogenic peptides as tumour vaccines. Anti-tumour active immunity primed by peptides from CD151 may be an effective and safe approach to suppress cancer progression.

High Yield Expression of Recombinant CD151 in E. coli and a Structural Insight into Cholesterol Binding Domain.

CD151 is an abundantly expressed eukaryotic transmembrane protein on the cell surface. It is involved in cell adhesion, angiogenesis and signal transduction as well in disease conditions such as cancer and viral infections. However, the molecular mechanism of CD151 activation is poorly understood due to the lack of structural information. By considering the difficulties in expressing the membrane protein in E. coli, herein we introduce the strategic design for the effective expression of recombinant CD151 protein in E. coli with high yield, that would aid for the structural studies. CD151 having four transmembrane domain (TMD's) along with small and a large extracellular loop (LEL) is constructed in parts to enhance the soluble expression of the protein attached with fusion tag. This has led to the high yield of the recombinant CD151 protein in the designed constructs. The recombinant CD151 protein is characterized and confirmed by western blot, CD and Mass peptide fingerprint. The molecular dynamics simulations (MDS) for the full-length CD151 shows conformational changes in the LEL of the protein in the presence and absence of cholesterol and indicate the certainty of closed and open conformation of CD151 based on cholesterol binding. The MDS results have led to the understanding of the possible underlying mechanism for the activation of the CD151 protein.

Tetraspanin CD151 and integrin α3ß1 contribute to the stabilization of integrin α6ß4-containing cell-matrix adhesions.

Tetraspanin CD151 has been suggested to regulate cell adhesion through its association with laminin-binding integrins α3ß1 and α6ß4; however, its precise function in keratinocyte adhesion remains elusive. In this study, we investigated the role of CD151 in the formation and maintenance of laminin-associated adhesions. We show that CD151, through binding to integrin α3ß1, plays a critical role in the stabilization of an adhesion structure with a distinct molecular composition of hemidesmosomes with tetraspanin features. These hybrid cell-matrix adhesions, which are formed early during cell adhesion and spreading and at later stages of cell spreading, are present in the central region of the cells. They contain the CD151-α3ß1/α6ß4 integrin complexes and the cytoskeletal linker protein plectin, but are not anchored to the keratin filaments. In contrast, hemidesmosomes, keratin filament-associated adhesions that contain integrin α6ß4, plectin, BP180 (encoded by COL17A1) and BP230 (encoded by DST), do not require CD151 for their formation or maintenance. These findings provide new insights into the dynamic and complex regulation of adhesion structures in keratinocytes and the pathogenic mechanisms underlying skin blistering diseases caused by mutations in the gene for CD151.

Reducing isoform complexity of human tetraspanins by optimized expression in Dictyostelium discoideum enables high-throughput functional read-out.

The human tetraspanin family of scaffold proteins comprises 33 isoforms. Being integral membrane proteins, they organize a so-called tetraspanin web via homomeric and heteromeric protein-protein interactions with integrins, immunoglobulins, growth factors, receptor tyrosine kinases, proteases, signaling proteins, and viral capsid proteins. Tetraspanins promote cellular effects, such as adhesion, migration, invasion, signaling, membrane fusion, protein trafficking, cancer progression, and infections. The ubiquitous expression of multiple tetraspanin isoforms and partner proteins hampers specific interaction studies. Here, we evaluated Dictyostelium discoideum as a non-mammalian expression system for human tetraspanins. Using high-content imaging we quantified tetraspanins in D. discoideum via fusion with green fluorescent protein. Three human tetraspanins, CD9, CD81, and CD151, served as test cases for which optimizations were carried out. We swapped the GFP domain between the N- and C-termini, added a Kozak sequence, and partially or fully adapted of the codon usage. This way, CD81 and CD151 were successfully produced. A conformation specific antibody further confirmed correct folding of CD81 and flow cytometry indicated an intracellular localization. Based on these data, we envision a D. discoideum-based co-expression platform with human partner proteins for studying tetraspanin interactions and their selective druggability on a large scale without the interference of endogenous human proteins.

Tetraspanins in infections by human cytomegalo- and papillomaviruses.

Members of the tetraspanin family have been identified as essential cellular membrane proteins in infectious diseases by nearly all types of pathogens. The present review highlights recently published data on the role of tetraspanin CD151, CD81, and CD63 and their interaction partners in host cell entry by human cytomegalo- and human papillomaviruses. Moreover, we discuss a model for tetraspanin assembly into trafficking platforms at the plasma membrane. These platforms might persist during intracellular viral trafficking.

The CD9, CD81, and CD151 EC2 domains bind to the classical RGD-binding site of integrin αvß3.

Tetraspanins play important roles in normal (e.g. cell adhesion, motility, activation, and proliferation) and pathological conditions (e.g. metastasis and viral infection). Tetraspanins interact with integrins and regulate integrin functions, but the specifics of tetraspanin-integrin interactions are unclear. Using co-immunoprecipitation with integrins as a sole method to detect interaction between integrins and full-length tetraspanins, it has been proposed that the variable region (helices D and E) of the extracellular-2 (EC2) domain of tetraspanins laterally associates with a non-ligand-binding site of integrins. We describe that, using adhesion assays, the EC2 domain of CD81, CD9, and CD151 bound to integrin αvß3, and this binding was suppressed by cRGDfV, a specific inhibitor of αvß3, and antibody 7E3, which is mapped to the ligand-binding site of ß3. We also present evidence that the specificity loop of ß3 directly bound to the EC2 domains. This suggests that the EC2 domains specifically bind to the classical ligand-binding site of αvß3. αvß3 was a more effective receptor for the EC2 domains than the previously known tetraspanin receptors α3ß1, α4ß1, and α6ß1. Docking simulation predicted that the helices A and B of CD81 EC2 bind to the RGD-binding site of αvß3. Substituting Lys residues at positions 116 and 144/148 of CD81 EC2 in the predicted integrin-binding interface reduced the binding of CD81 EC2 to αvß3, consistent with the docking model. These findings suggest that, in contrast with previous models, the ligand-binding site of integrin αvß3, a new tetraspanin receptor, binds to the constant region (helices A and B) of the EC2 domain.

α3ß1 integrins regulate CD151 complex assembly and membrane dynamics in carcinoma cells within 3D environments.

Integrins are extracellular matrix (ECM) receptors that are key players in the regulation of tumour cell invasion. The laminin-binding integrin α3ß1 has previously been shown to regulate adhesion and migration of carcinoma cells in part through co-operative signalling with the tetraspanin family of transmembrane proteins. However, the spatial and temporal regulation of crosstalk between these families of transmembrane proteins in intact cells remains poorly understood. Here we have used fluorescence resonance energy transfer (FRET) to demonstrate for the first time that α3ß1 and the tetraspanin CD151 directly associate at the front and retracting rear of polarised migrating breast carcinoma cells in both two-dimentional (2D) and three-dimentional (3D)matrices. Furthermore, localised α3ß1-CD151 binding correlates with lower CD151 homodimerisation in cells migrating on laminin or within matrigel. Loss of α3ß1 integrin leads to increased CD151 homodimer formation, increased activation of Rho GTPase, loss of cell polarity and decreased invasion in 3D ECM. As a result, α3-silenced cells show decreased actin-based membrane protrusion and retraction in both 2D and 3D environments. These data demonstrate that associations between α3ß1 and CD151 occur dynamically within discrete subcellular compartments and act to establish local GTPase signalling to promote tumour cell invasion. These novel findings shed light on the complex crosstalk and switching between receptor complexes in response to different extracellular cues during cell invasion in 3D environments.