Interaction between Yersinia pestis Ail Outer Membrane Protein and the C-Terminal Domain of Human Vitronectin.

Yersinia pestis, the causative agent of plague, is capable of evading the human immune system response by recruiting the plasma circulating vitronectin proteins, which act as a shield and avoid its lysis. Vitronectin recruitment is mediated by its interaction with the bacterial transmembrane protein Ail, protruding from the Y. pestis outer membrane. By using all-atom long-scale molecular dynamic simulations of Ail embedded in a realistic model of the bacterial membrane, we have shown that vitronectin forms a stable complex, mediated by interactions between the disordered moieties of the two proteins. The main amino acids driving the complexation have also been evidenced, thus favoring the possible rational design of specific peptides which, by inhibiting vitronectin recruitment, could act as original antibacterial agents.

Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

Potential Benefits of Integrin αvß3 Antagonists in a Mouse Model of Experimental Dry Eye.

PURPOSE: The purpose of this study was to extensively evaluate the efficacy of integrin αvß3 antagonists for the treatment of experimental dry eye (EDE). METHODS: Vitronectin, an αvß3 ligand, was used to induce tumor necrosis factor-α gene expression in human THP-1 macrophages. To induce EDE, C57BL/6 mice were housed in a low-humidity controlled environment chamber and injected subcutaneously with scopolamine for 7 days. Subsequently, αvß3 antagonists, including RGDfD, c(RGDfD), c(RGDiD), c(RGDfK), ATN-161, SB273005, and cilengitide, were administered topically to EDE animals under controlled environment chamber conditions. Corneal epithelial damage in EDE was assessed by fluorescein staining. The density of conjunctival goblet cells and secretion of tears was measured by period acid-Schiff staining and phenol red-impregnated cotton threads, respectively. Inflammation markers, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-17A, and metalloproteinase (MMP)-9, in the pooled cornea and conjunctiva tissues were examined by real-time polymerase chain reaction. RESULTS: The inhibitory effects of αvß3 antagonists on the vitronectin-induced tumor necrosis factor-α gene expression and integrin-mediated inflammatory signaling were validated in THP-1 macrophages. αvß3 antagonists ameliorated the impairment of the corneal epithelial barrier with varying therapeutic efficacies, compared with vehicle-treated mice. c(RGDfD) and c(RGDiD) significantly protected against goblet cell loss, tear reduction, and proinflammatory gene expression in EDE. CONCLUSIONS: Topical applications of αvß3 antagonists yield therapeutic benefits in EDE by promoting corneal epithelial defect healing and reducing inflammation. Antagonistic targeting αvß3 may be a novel promising strategy to treat patients with dry eye disease.

Macrophage promotes fibroblast activation and kidney fibrosis by assembling a vitronectin-enriched microenvironment.

Background: Renal infiltration of inflammatory cells including macrophages is a crucial event in kidney fibrogenesis. However, how macrophage regulates fibroblast activation in the fibrotic kidney remains elusive. In this study, we show that macrophages promoted fibroblast activation by assembling a vitronectin (Vtn)-enriched, extracellular microenvironment. Methods: We prepared decellularized kidney tissue scaffold (KTS) from normal and fibrotic kidney after unilateral ischemia-reperfusion injury (UIRI) and carried out an unbiased quantitative proteomics analysis. NRK-49F cells were seeded on macrophage-derived extracellular matrix (ECM) scaffold. Genetic Vtn knockout (Vtn-/-) mice and chronic kidney disease (CKD) model with overexpression of Vtn were used to corroborate a role of Vtn/integrin αvß5/Src in kidney fibrosis. Results: Vtn was identified as one of the most upregulated proteins in the decellularized kidney tissue scaffold from fibrotic kidney by mass spectrometry. Furthermore, Vtn was upregulated in the kidney of mouse models of CKD and primarily expressed and secreted by activated macrophages. Urinary Vtn levels were elevated in CKD patients and inversely correlated with kidney function. Genetic ablation or knockdown of Vtn protected mice from developing kidney fibrosis after injury. Conversely, overexpression of Vtn exacerbated renal fibrotic lesions and aggravated renal insufficiency. We found that macrophage-derived, Vtn-enriched extracellular matrix scaffold promoted fibroblast activation and proliferation. In vitro, Vtn triggered fibroblast activation by stimulating integrin αvß5 and Src kinase signaling. Either blockade of αvß5 with neutralizing antibody or pharmacological inhibition of Src by Saracatinib abolished Vtn-induced fibroblast activation. Moreover, Saracatinib dose-dependently ameliorated Vtn-induced kidney fibrosis in vivo. These results demonstrate that macrophage induces fibroblast activation by assembling a Vtn-enriched extracellular microenvironment, which triggers integrin αvß5 and Src kinase signaling. Conclusion: Our findings uncover a novel mechanism by which macrophages contribute to kidney fibrosis via assembling a Vtn-enriched extracellular niche and suggest that disrupting fibrogenic microenvironment could be a therapeutic strategy for fibrotic CKD.

Periosteal Bone Formation Varies with Age in Periostin Null Mice.

Periostin, also known as osteoblast-specific factor 2, is a matricellular protein predominantly expressed at the periosteum of bone. During growth and development, periostin contributes to periosteal expansion by facilitating osteoblast differentiation and mineralization. Later in life, periosteal expansion provides an adaptive strategy to increase tissue strength without requiring substantial increase in bone mass. However, the function of periostin past skeletal maturity and during advanced aging is relatively unknown. The objective of this study was to examine the function of periostin in maintaining bone mass and tissue strength across different ages. In periostin null mice (Postn-/-), periosteal bone formation was significantly reduced in young (3 months) and adult mice (9 months). The lack of bone formation resulted in reduced bone mass and ultimate strength. Conversely, periosteal bone formation increased at advanced ages in 18-month-old Postn-/- mice. The increase in periosteal mineralization at advanced ages coincides with increased expression of vitronectin and osteopontin. Periosteal progenitors from Postn-/- mice displayed an increased capacity to mineralize when cultured on vitronectin, but not type-1 collagen. Altogether, these findings demonstrate the unique role of periostin in regulating periosteal bone formation at different ages and the potential for vitronectin to compensate in the absence of periostin.

Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions.

To improve mesenchymal stem cell (MSC)-based therapy efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under microenvironmental stress conditions. We observed that human Wharton's jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-κß was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, formed under serum starvation stress. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. VTN is established as a key player which possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.

Knockdown of FBLN2 suppresses TGF-ß1-induced MRC-5 cell migration and fibrosis by downregulating VTN.

Idiopathic pulmonary fibrosis (IPF) is a common chronic and progressive lung disease. Fibulin-2 (FBLN2) is upregulated in patients with IPF; however, its exact role in IPF remains unclear. The present study aimed to investigate the role and the regulatory mechanism of FBLN2 in TGF-ß1-induced fibrogenesis using human lung fibroblast-derived MRC-5 cells. Cell transfection was performed to regulate FBLN2 expression. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the expression levels of FBLN2 and vitronectin (VTN). Cell viability and migration were determined via the Cell Counting Kit-8 and wound healing assays, respectively. Immunofluorescence was performed to detect α-smooth muscle actin (α-SMA)-positive cells. The STRING database was used to predict the interaction between FBLN2 and VTN, which was verified via the protein immunoprecipitation assay. The results demonstrated that inhibition of FBLN2 notably inhibited TGF-ß1-induced proliferation and migration, as well as downregulating the protein expression levels of MMP2 and MMP9 in MRC-5 cells. In addition, inhibition of FBLN2 suppressed the expression levels of α-SMA, collagen type 1 α1 and fibronectin. FBLN2 was demonstrated to bind to VTN and negatively regulate its expression. Furthermore, overexpression of VTN partly abolished the inhibitory effects of FBLN2 knockdown on TGF-ß1-induced proliferation, migration and fibrosis, as well as the activity of focal adhesion kinase (FAK) signaling. Taken together, the results of the present study suggest that FBLN2 knockdown can attenuate TGF-ß1-induced fibrosis in MRC-5 cells by downregulating VTN expression via FAK signaling. Thus, FBLN2 may be a potential therapeutic target for IPF treatment.

Selective Grafting of Protease-Resistant Adhesive Peptides on Titanium Surfaces.

In orthopedic, dental, and maxillofacial fields, joint prostheses, plates, and screws are widely used in the treatment of problems related to bone tissue. However, the use of these prosthetic systems is not free from complications: the fibrotic encapsulation of endosseous implants often prevents optimal integration of the prostheses with the surrounding bone. To overcome these issues, biomimetic titanium implants have been developed where synthetic peptides have been selectively grafted on titanium surfaces via Schiff base formation. We used the retro-inverted sequence (DHVPX) from [351-359] human Vitronectin and its dimer (D2HVP). Both protease-resistant peptides showed increased human osteoblast adhesion and proliferation, an augmented number of focal adhesions, and cellular spreading with respect to the control. D2HVP-grafted samples significantly enhance Secreted Phosphoprotein 1, Integrin Binding Sialoprotein, and Vitronectin gene expression vs. control. An estimation of peptide surface density was determined by Two-photon microscopy analysis on a silanized glass model surface labeled with a fluorescent analog.

A Vitronectin-Derived Peptide Restores Ovariectomy-Induced Bone Loss by Dual Regulation of Bone Remodeling.

BACKGROUND: Bone remodeling is tightly regulated through bone resorption and bone formation; imbalances in bone remodeling can cause various pathological conditions such as osteoporosis. Antiresorptive agents commonly used for treating osteoporosis do not substantially reverse osteoporotic bone loss. METHODS: We evaluated the effects of the RVYFFKGKQYWE motif (residues 270-281; VnP-16) of human vitronectin on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoclastogenesis of bone marrow-derived macrophages. The effects of VnP-16 were also assessed in a mouse model of estrogen deficiency-induced osteoporosis (ovariectomized female C57BL/6 mice). To assay whether VnP-16 can reverse ovariectomy-induced bone loss, synthetic peptides or vehicle were subcutaneously injected into ovariectomized mice once a week for 4 weeks (n = 10/group). To evaluate the bone restorative effects of VnP-16, in-vivo micro-computed tomography analysis and histological staining were performed. RESULTS: VnP-16 induced osteogenic differentiation of hMSCs and inhibited the RANKL-RANK-TRAF6 axis in the osteoclastogenesis signaling pathway. Furthermore, systemic administration of VnP-16 reversed ovariectomy-induced bone loss in the femoral neck, distal femur and lumbar spine by increasing osteoblast differentiation and promoting bone formation, and concomitantly decreasing osteoclastogenesis and inhibiting bone resorption. The bone restorative effect of VnP-16 was observed one week after subcutaneous administration, and although the timing of the effect differed according to bone location, it persisted for at least 3 weeks. CONCLUSION: Our findings suggest that VnP-16 is a potential therapeutic agent for treating osteoporosis that mediates its effects through dual regulation of bone remodeling.

Role of Vitronectin and Its Receptors in Neuronal Function and Neurodegenerative Diseases.

Vitronectin (VTN), a multifunctional glycoprotein with various physiological functions, exists in plasma and the extracellular matrix. It is known to be involved in the cell attachment, spreading and migration through binding to the integrin receptor, mainly via the RGD sequence. VTN is also widely used in the maintenance and expansion of pluripotent stem cells, but its effects go beyond that. Recent evidence shows more functions of VTN in the nervous system as it participates in neural differentiation, neuronutrition and neurogenesis, as well as in regulating axon size, supporting and guiding neurite extension. Furthermore, VTN was proved to play a key role in protecting the brain as it can reduce the permeability of the blood-brain barrier by interacting with integrin receptors in vascular endothelial cells. Moreover, evidence suggests that VTN is associated with neurodegenerative diseases, such as Alzheimer's disease, but its function has not been fully understood. This review summarizes the functions of VTN and its receptors in neurons and describes the role of VTN in the blood-brain barrier and neurodegenerative diseases.

Potential Urine Proteomic Biomarkers for Focal Segmental Glomerulosclerosis and Minimal Change Disease.

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.

Calcium-induced environmental adaptability of the blood protein vitronectin.

The adaptability of proteins to their work environments is fundamental for cellular life. Here, we describe how the hemopexin-like domain of the multifunctional blood glycoprotein vitronectin binds Ca2+ to adapt to excursions of temperature and shear stress. Using X-ray crystallography, molecular dynamics simulations, NMR, and differential scanning fluorimetry, we describe how Ca2+ and its flexible hydration shell enable the protein to perform conformational changes that relay beyond the calcium-binding site and alter the number of polar contacts to enhance conformational stability. By means of mutagenesis, we identify key residues that cooperate with Ca2+ to promote protein stability, and we show that calcium association confers protection against shear stress, a property that is advantageous for proteins that circulate in the vasculature, like vitronectin.

Hsa_circ_0079530/AQP4 Axis Is Related to Non-Small Cell Lung Cancer Development and Radiosensitivity.

BACKGROUND: Circular RNAs are associated with non-small cell lung cancer (NSCLC) development and radiosensitivity. Nevertheless, the function and regulation mechanism of hsa_circ_0079530 (circ_0079530) in NSCLC development and radiosensitivity are largely unknown. METHODS: The abundances of circ_0079530, microRNA (miR-409-3p), aquaporin 4 (AQP4), E-cadherin, intercellular adhesion molecule-1, vitronectin, proliferating cell nuclear antigen, and matrix metalloproteinase 9 were determined via quantitative reverse transcription polymerase chain reaction or western blotting. Cell proliferation, survival fraction, cycle process, migration, invasion, and in vivo growth were examined by cell counting kit-8, colony formation, flow cytometry, transwell, and xenograft analyses. The binding relationship was assessed via dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: Circ_0079530 expression was increased in NSCLC tissues and radioresistant samples. Circ_0079530 knockdown restrained cell proliferation, migration, and invasion, and facilitated radiosensitivity. Circ_0079530 silence decreased tumor growth with or without radiation treatment. Circ_0079530 was verified as a miR-409-3p sponge, and miR-409-3p downregulation mitigated the effects of circ_0079530 interference on NSCLC cell malignancy and radiosensitivity. AQP4 was directly targeted by miR-409-3p. MiR-409-3p restrained cell proliferation, migration, and invasion, and enhanced radiosensitivity by decreasing AQP4 expression. Notably, circ_0079530 silence decreased AQP4 expression by regulating miR-409-3p expression. CONCLUSION: Circ_0079530 silence repressed cell proliferation, migration, and invasion, and facilitated radiosensitivity in NSCLC cells by mediating miR-409-3p/AQP4 axis.

Female-specific neuroprotection after ischemic stroke by vitronectin-focal adhesion kinase inhibition.

We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40-45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN-/- females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.

A vitronectin-derived dimeric peptide suppresses osteoclastogenesis by binding to c-Fms and inhibiting M-CSF signaling.

Vitronectin is an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bone, and is involved in diverse physiological processes. Here, we developed a new bioactive dimeric peptide (VnP-8-DN1 dimer) from a human vitronectin-derived motif (IDAAFTRINCQG; residues 206-217; VnP-8) via removal of an isoleucine residue at the N-terminus of VnP-8 and spontaneous air oxidation. The VnP-8-DN1 dimer potently enhanced cell attachment activity, and this activity was mediated by binding to cellular heparan sulfate proteoglycan receptors. Moreover, the VnP-8-DN1 dimer suppressed osteoclast differentiation by blocking the early stage of osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Furthermore, the VnP-8-DN1 dimer decreased the bone-resorbing activity of osteoclasts and increased the survival of osteoclast precursor cells by decreasing the cellular level of c-Fms and reducing RANK expression. Taken together, these results demonstrate that the VnP-8-DN1 dimer inhibits the early stages of M-CSF- and RANK-induced osteoclast differentiation by binding to c-Fms and inhibiting M-CSF signaling.

Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier.

Endothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surfaces of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, α5, in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding, as well as endothelial-specific deletion of integrin α5, causes barrier leakage in mice. Furthermore, vitronectin-integrin α5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.

Extracellular matrix modulates T cell clearance of malignant cells in vitro.

Despite the success of T cell checkpoint therapies, breast cancers rarely express these immunotherapy markers and are believed to be largely "immune cold" with limited inflammation and immune activation. The reason for this limited immune activation remains poorly understood. We sought to determine whether extracellular matrix substrate could contribute to this limited immune activation. Specifically, we asked whether extracellular matrix could alter T cell cytotoxicity against malignant mammary gland carcinoma cells (MCC) in a setup designed to promote maximal T cell efficacy (i.e., rich media with abundant IL2, high ratio of T cells to MCC). We observed that T cell clearance of MCC varied from 0% in collagen 4 or 6 conditions to almost 100% in fibronectin or vitronectin. Transcriptomics revealed that T cell function was defective in MCC/T cell cocultures on collagen 4 (Col4), potentially corresponding to greater expression of cytokines MCC cultured in this environment. In contrast, transcriptomics revealed an effective, exhausted phenotype on vitronectin. The observation that Col4 induces T cell suppression suggests that targeting tumor-ECM interactions may permit new approaches for utilizing immunotherapy in tumors which do not provoke a strong immune response.

The effect of endocrine disrupting chemicals on the vitronectin-receptor (integrin αvß3)-mediated cell adhesion of human umbilical vein endothelial cells.

Endocrine disrupting chemicals (EDCs) are associated with cancer development and progression due to their promotion of increased cell invasiveness and metastasis formation. However, the effects of EDCs on cell adhesion mediated through integrins have not been well studied to date. Their actions are implicated by binding sites for hormones on the vitronectin receptor (VTNR; or integrin αvß3), which is involved in tumor angiogenesis and metastasis. VTNR-expressing human umbilical vein endothelial cells (HUVECs) were used to determine the effects of EDCs and endogenous hormones on cell adhesion to vitronectin-coated surfaces, and on VTNR activation. Cell adhesion was significantly increased for bisphenol A, triclocarban, and triclosan (10, 100 nM; p < 0.05), with similar trends for bisphenols AF and S (10, 100 nM; p > 0.05). No changes in cell adhesion were seen for 5α-dihydrotestosterone, 17ß-estradiol, triiodothyronine, imatinib and paroxetine. These data indicate that EDC-mediated increases in HUVEC adhesion to vitronectin are not mediated through androgenic, estrogenic, or thyroid activities, nor through activation of VTNR. Although these effects of EDCs on HUVEC adhesion require further investigation of the underlying mechanism(s) of action to define their biological relevance, the low-dose effects and nonmonotonic responses revealed here define the need for further investigation of these EDCs.

Towards understanding the novel adhesin function of Candida albicans phosphoglycerate mutase at the pathogen cell surface: some structural analysis of the interactions with human host extracellular matrix proteins.

Although many atypical proteinaceous cell wall components that belong to a group of multitasking, "moonlighting" proteins, have been repeatedly identified in numerous pathogenic microorganisms, their novel extracellular functions and secretion mechanisms remain largely unrecognized. In Candida albicans, one of the most common fungal pathogens in humans, phosphoglycerate mutase (Gpm1) - a cytoplasmic enzyme involved in the glycolysis pathway - has been shown to occur on the cell surface and has been identified as a potentially important virulence factor. In this study, we demonstrated tight binding of C. albicans Gpm1 to the candidal cell surface, thus suggesting that the readsorption of soluble Gpm1 from the external environment could be a likely mechanism leading to the presence of this moonlighting protein on the pathogen surface. Several putative Gpm1-binding receptors on the yeast surface were identified. The affinities of Gpm1 to human vitronectin (VTR) and fibronectin (FN) were characterized with surface plasmon resonance measurements, and the dissociation constants of the complexes formed were determined to be in the order of 10-8 M. The internal Gpm1 sequence motifs, directly interacting with VTR (aa 116-158) and FN (aa 138-175) were mapped using chemical crosslinking and mass spectrometry. Synthetic peptides with matching sequences significantly inhibited formation of the Gpm1-VTR and Gpm1-FN complexes. A molecular model of the Gpm1-VTR complex was developed. These results provide the first structural insights into the adhesin function of candidal surface-exposed Gpm1.