Biosynthesis of prostaglandins

Highly purified cyclooxygenase from sheep vesicular glands is stimulated by the presence of protoporphyrin IX compounds. This stimulation may be due to the conversion of an apoenzyme to the holoenzyme, and full activity is achieved when half of the enzyme subunits (70,000 daltons) bind heme. Also, one‐half of the subunits appear to contain non‐heme iron. The apparent molecular weight of the holoenzyme is approximately 300,000 daltons and is compatible with a complex of four 70,000 dalton subunits. Thus, we suggest that heme and non‐heme iron may be attached to different 70,000 daltons subunits that make up an A2B2‐type of peptide chain arrangement.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141216/1/lipd0591.pd

EWI-2 regulates α3β1 integrin–dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (α2β1 integrin ligand). However, on laminin-5 (α3β1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to α3β1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced α3β1–CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance α3β1–CD81 complex formation. These results show how laterally associated EWI-2 might regulate α3β1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes

Structural organization and interactions of transmembrane domains in tetraspanin proteins

BACKGROUND: Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. RESULTS: Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)(n). In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. CONCLUSION: Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues

Accelerative autoactivation of prostaglandin biosynthesis by PGG2

Cyclooxygenase catalysis is stimulated by its product, PGG2, and by other lipid hydroperoxides. The endoperoxide, PGH2, was not stimulatory. The results provide a direct demonstration of an essential role for lipid hydroperoxides in prostaglandin biosynthesis, and show how the biosynthetic intermediate PGG2 has a positive accelerative effect.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22460/1/0000001.pd

Palmitoylation supports assembly and function of integrin–tetraspanin complexes

As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (α3, α6, and β4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of β4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient β4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core α6β4–CD151 complex formation was unaltered. There is also a functional connection between CD9 and β4 integrins, as evidenced by anti-CD9 antibody effects on β4-dependent cell spreading. Notably, β4 palmitoylation neither increased localization into “light membrane” fractions of sucrose gradients nor decreased solubility in nonionic detergents—hence it does not promote lipid raft association. Instead, palmitoylation of β4 (and of the closely associated tetraspanin CD151) promotes CD151–α6β4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation

Synthesis of (8Z,14Z)-13,13-dimethyleicosa-8,14-dien-11-ynoic acid as an inhibitor of prostaglandin cyclooxygenase

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23081/1/0000656.pd

Tetraspanin CD151 plays a key role in skin squamous cell carcinoma

Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size, and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared to other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3, a regulator of cell proliferation and apoptosis. CD151 also supports PKCα-α6β4 integrin association and PKC-dependent β4 S1424 phosphorylation, while regulating α6β4 distribution. CD151-PKCα effects on integrin β4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including DMBA (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting EGFR, PKC, Jak2/Tyk2, and STAT3. Hence, CD151 ‘co-targeting’ may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151-laminin-binding integrin complexes