Designed synthetic analogs of the α‐helical peptide temporin‐La with improved antitumor efficacies via charge modification and incorporation of the integrin αvβ3 homing domain

How to target cancer cells with high specificity and kill cancer cells with high efficiency remains an urgent demand for anticancer drugs. Temporin‐La, which belongs to the family of temporins, presents antitumor activity against many cancer cell lines. We first used a whole bioinformatic analysis method as a platform to identify new anticancer antimicrobial peptides (AMPs). On the basis of these results, we designed a temporin‐La analog (temporin‐Las) and related constructs containing the Arg‐Gly‐Asp (RGD) tripeptide, the integrin αvβ3 homing domain (RGD‐La and RGD‐Las). We detected a link between the net charges and integrin αvβ3 expression of cancer cell lines and the antitumor activities of these peptides. Temporin‐La and its synthetic analogs inhibited cancer cell proliferation in a dose‐dependent manner. Evidence was provided that the affinity between RGD‐Las and tumor cell membranes was stronger than other tested peptides using a pull‐down assay. Morphological changes on the cell membrane induced by temporin‐La and RDG‐Las, respectively, were examined by scanning electron microscopy. Additionally, time‐dependent morphological changes were detected by confocal microscopy, where the binding process of RGD‐Las to the cell membrane could be monitored. The results indicate that the electrostatic interaction between these cationic peptides and the anionic cell membrane is a major determinant of selective cell killing. Thus, the RGD tripeptide is a valuable ligand motif for tumor targeting, which leads to an increased anticancer efficiency by RGD‐Las. These AMP‐derived peptides have clinical potential as specifically targeting agents for the treatment of αvβ3 positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Activation mechanisms of αVβ3 integrin by binding to fibronectin: A computational study

Integrin αVβ3 plays an important role in regulating cellular activities and in human diseases. Although the structure of αVβ3 has been studied by crystallography and electron microscopy, the detailed activation mechanism of integrin αVβ3 induced by fibronectin remains unclear. In this study, we investigated the conformational and dynamical motion changes of Mn²⁺‐bound integrin αVβ3 by binding to fibronectin with molecular dynamics simulations. Results showed that fibronectin binding to integrin αVβ3 caused the changes of the conformational flexibility of αVβ3 domains, the essential mode of motion for the domains of αV subunit and β3 subunit and the degrees of correlated motion of residues between the domains of αV subunit and β3 subunit of integrin αVβ3. The angle of Propeller domain with respect to the Calf‐2 domain of αV subunit and the angle of Hybrid domain with respect to βA domain of β3 subunit significantly increased when integrin αVβ3 was bound to fibronectin. These changes could result in the conformational change tendency of αVβ3 from a bend conformation to an extended conformation and lead to the open swing of Hybrid domain relative to βA domain of β3 subunit, which have demonstrated their importance for αVβ3 activation. Fibronectin binding to integrin αVβ3 significantly decreased the relative position of α1 helix to βA domain and that to metal ion‐dependent adhesion site, stabilized Mn²⁺ ions binding in integrin αVβ3 and changed fibronectin conformation, which are important for αVβ3 activation. Results from this study provide important molecular insight into the “outside‐in” activation mechanism of integrin αVβ3 by binding to fibronectin.     

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSP-RGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors. J. Cell. Physiol. 176:482–494, 1998. © 1998 Wiley-Liss, Inc.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Protein interactions with the platelet integrin αIIb regulatory motif

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin α‐subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet‐specific integrin α_IIbβ₃. We employ peptide‐affinity chromatography followed‐up with LC‐MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR‐peptide. Of these, seven have been reported in the PPI literature as interactors with integrin α‐subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin‐tagged α‐integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide‐affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

Phorbol myristate acetate transactivates the avian β3 integrin gene and induces αvβ3 integrin expression

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) transactivates the avian β₃ integrin gene whose promoter contains at least two vitamin D response elements, one of which is in close proximity to a candidate AP1 site (TGACTCA). Since fos/jun and steroid hormones interact to regulate gene expression, we asked whether phorbol-12-myristate-13-acetate (PMA), which stimulates binding of fos/jun to AP1 sites, transactivates the avian β₃ integrin gene and, if so, does the phorbol ester modulate 1,25(OH)₂D₃ induction of the gene. We find the candidate AP1 sequence comigrates with the consensus AP1 sequence on electromobility shift assay when incubated with recombinant c-jun protein. Furthermore, PMA prompts expression of β₃ integrin mRNA in the avian monocytic line, HD11. The increase in message reflects transactivation of the β₃ gene and is mirrored by plasma membrane appearance of the integrin heterodimer α_vβ₃. Moreover, attesting to the functional significance of PMA-enhanced α_vβ₃ expression, cells treated with concentrations of the phorbol ester that induce the β₃ gene, spread extensively on plastic, an event blocked by an anti-α_v antibody and a peptide mimetic known to inhibit α_vβ₃-mediated cell attachment. Interestingly, co-addition of 1.25(OH)₂D₃ and PMA prompts greater expression of α_vβ₃ than when the cells are exposed to either agent alone and PMA enhances 1,25(OH)₂D₃-induced β₃ integrin mRNA expression. Thus, PMA and 1,25(OH)₂D₃ impact on the avian β₃ integrin gene independently and in combination. © 1996 Wiley-Liss, Inc.     

Osteopontin mediates macrophage chemotaxis via α4 and α9 integrins and survival via the α4 integrin

Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis and the foreign body reaction. However, the molecular mechanism behind OPN regulation of macrophage functions is not well understood. OPN is a secreted molecule and interacts with several integrins via two domains: the RGD sequence binding to α_v‐containing integrins, and the SLAYGLR sequence binding to α₄β₁, α₄β₇, and α₉β₁ integrins. Here we determined the role of OPN in macrophage survival, chemotaxis, and activation state. For survival studies, OPN treated‐bone marrow derived macrophages (BMDMs) were challenged with growth factor withdrawal and neutralizing integrin antibodies. We found that survival in BMDMs is mediated primarily through the α₄ integrin. In chemotaxis studies, we observed that migration to OPN was blocked by neutralizing α₄ and α₉ integrin antibodies. Further, OPN did not affect macrophage activation as measured by IL‐12 production. Finally, the relative contributions of the RGD and the SLAYGLR functional domains of OPN to leukocyte recruitment were evaluated in an in vivo model. We generated chimeric mice expressing mutated forms of OPN in myeloid‐derived leukocytes, and found that the SLAYGLR functional domain of OPN, but not the RGD, mediates macrophage accumulation in response to thioglycollate‐elicited peritonitis. Collectively, these data indicate that α₄ and α₉ integrins interacting with OPN via the SLAYGLR domain play a key role in macrophage biology by regulating migration, survival, and accumulation. J. Cell. Biochem. 114: 1194–1202, 2013. © 2012 Wiley Periodicals, Inc.     

CTGF enhances migration and MMP‐13 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CTGF increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody (mAb) and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the CTGF‐induced increase of the migration and MMP‐13 up‐regulation of chondrosarcoma cells. CTGF stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited CTGF‐induced cell migration and MMP‐13 up‐regulation. Stimulation of JJ012 cells with CTGF also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The CTGF‐mediated increases in κB‐luciferase activities were inhibited by RGD, PD98059, U0126 or FAK, and ERK2 mutant. Taken together, our results indicated that CTGF enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3 integrin, FAK, ERK, and NF‐κB signal transduction pathway. J. Cell. Biochem. 107: 345–356, 2009. © 2009 Wiley‐Liss, Inc.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Reversing microtubule‐directed chemotherapeutic drug resistance by co‐delivering α2β1 inhibitor and paclitaxel with nanoparticles in ovarian cancer

Previous reports indicated that integrins associated signals are tightly related to tumor progression. Here, we observed elevated expression of integrin α2β1 in tumor tissues from microtubule‐directed chemotherapeutic drugs (MDCDs) resistant patients compared with the samples from chemosensitive patients. More importantly, we sorted the integrin α2β1⁺ tumor cells and found those cells revealed high MDCDs resistance, whereas MDCDs shows effective cytotoxicity to those integrin α2β1^? tumor cells in vitro and in vivo. Mechanistically, we demonstrated that integrin α2β1 could induce MDCDs resistance through the activation of the PI3K/AKT pathway. Applying MPEG‐PLA to co‐encapsulate the integrin α2β1 inhibitor E7820 and MDCDs could effectively reverse MDCDs resistance, resulting in enhanced anticancer effects while avoiding potential systemic toxicity in vitro and in vivo. In conclusion, the expression of integrin α2β1 contributes to MDCDs resistance, while applying E7820 combination treatment by MPEG‐PLA nanoparticles could reverse the resistance.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Signalling mechanisms of the leukocyte integrin αMβ2: Current and future perspectives

Integrins are a family of heterodimeric cell adhesion receptors expressed on most cells and are involved in many cellular functions including phagocytosis, a process by which professional phagocytes recognise, bind and internalise foreign materials larger than 0.5 µm in diameter. An example of a phagocytic integrin receptor is αMβ2, and this review seeks to provide fresh insights into the current knowledge of this subject. Key areas that this review will emphasise include, the classical understanding of bi‐directional signalling to and from αMβ2 (aka inside‐out and outside‐in signalling, respectively). For inside‐out signalling, we will review the involvement of the small GTPase, Rap1, FERM‐containing proteins such as talin and kindlin‐3, some of the kinases, and the GEF, cytohesin‐1 and vasodilator‐stimulated phosphoprotein (VASP). We also summarise studies into outside‐in signalling, focussing on the roles of RhoA and RhoG, and activation of Rac1 through the complex comprising TIAM, 14‐3‐3 and β2. We will also consider non‐classical signalling processes, which include integrin clustering and membrane ruffling. Through this review, we hope to highlight the importance of αMβ2 signalling mechanisms and their relevance to other integrin‐mediated events.     

His‐tag binding by antibody C706 mimics β‐amyloid recognition

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ₄₂ peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2₁ and C2. Although the Fab was crystallized in the presence of Aβ₁₆, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.