28 research outputs found
Sphingosine-1-phosphate inhibits vascular smooth muscle cell migration by activating GĪ±āā and RhoA
Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to [email protected], referencing the URI of the item.Includes bibliographical references (leaves 24-27).Angiogenesis, the formation of new blood vessels, is important in growth, healing, and disease. Sphingosine-1-phosphate (S1P) is a lysophospholipid with potent angiogenic effects. One of these effects is its regulation of cellular migration, an early step in angiogenesis. S1P exerts contradictory effects on different types of vascular cells, strongly inducing endothelial cell (EC) migration while strongly inhibiting vascular smooth muscle cells (VSMC) migration. Here, we demonstrate that S1P inhibits the migration of VSMCs by activating G[Ī±]āā and RhoA. S1P abolished the migration of VSMCs in response to platelet-derived growth factor (PDGF), a potent stimulator of VSMC migration. PDGF strongly induced the formation of lamellipodia, which are membrane ruffles associated with cell migration. S1P eliminated these and induced the formation of stress fibers, which are actin cytoskeletal structures that stabilize the cell. The members of the Rho family of small GTPases regulate cytoskeletal morphology and function. Rac1 induces lamellipodia, RhoA induces stress fibers, and Cdc42 induces filopodia, which are small finger-like membrane spikes. We introduced dominant-negative (DN) and constitutively active (CA) mutants of the Rho GTPases into VSMCs using adenoviral vectors. The results indicated that S1P inhibits VSMC migration by activating RhoA. RhoA activation was also demonstrated directly using a RhoA pull-down assay. S1P exerts its cellular effects by activating the endothelial differentiation gene G-protein-coupled receptors, which initiate signaling pathways by activating different G[Ī±] subunits of heterotrimeric G proteins. To determine which G[Ī±] subunit S1P activates in order to activate RhoA, we introduced DN and CA mutants of G[Ī±]āā and G[Ī±]āā into VSMCs through adenoviral vectors. The results indicated that S1P activates RhoA by activating G[Ī±]āā. These results are important because they further elucidate some of the key signaling pathways regulating angiogenesis. When angiogenesis begins, ECs migrate in response to S1P and other factors. Interestingly, our new data suggest that S1P may prevent VSMCs from interfering with early EC tube assembly, by inhibiting VSMC migration through selective activation of G[Ī±]āā and RhoA. Later, after the ECs have established a network of capillary tubes, they secrete additional factors, such as PDGF, which attract VSMCs to them, allowing mature blood vessels to form
Advancing Science through Conversations: Bridging the Gap between Blogs and the Academy
Blogs have stormed the Internet, providing an interactive medium for rapid and wide-reaching information dispersal. But is there a place for blogs in academia
Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling
BACKGROUND: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D) and invasion of three-dimensional (3D) collagen matrices and, in particular, evaluated the role of MT1-MMP in this process. RESULTS: LPA stimulated while S1P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (~700 Ī¼m over 48 hours) in response to either lipid. siRNA targeting of LPA(1 )and Rac1, or S1P(1), Rac1, and Cdc42 specifically inhibited LPA- or S1P-induced HT1080 invasion, respectively. Analysis of LPA-induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (~125 Ī¼m vs. ~45 Ī¼m) and velocity of invasion (~0.09 Ī¼m/min vs. ~0.03 Ī¼m/min) only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs)-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT)-MMPs. Furthermore, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process. CONCLUSION: LPA is a fundamental regulator of MT1-MMP-dependent tumor cell invasion of 3D collagen matrices. In contrast, S1P appears to act as an inhibitory stimulus in most cases, while stimulating only select tumor lines. MT1-MMP is required only when tumor cells navigate 3D barriers and not when cells migrate on 2D substrata. We demonstrate that tumor cells require coordinate regulation of LPA/S1P receptors and Rho GTPases to migrate, and additionally, require MT1-MMP in order to invade collagen matrices during neoplastic progression
A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data
The measurement of 1H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize
sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metalchelating
paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel,
disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed Ī±-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed Ī±-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label
Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3
The endothelial cell (EC)āderived tissue inhibitor of metalloproteinase-2 (TIMP-2) and pericyte-derived TIMP-3 are shown to coregulate human capillary tube stabilization following ECāpericyte interactions through a combined ability to block EC tube morphogenesis and regression in three-dimensional collagen matrices. ECāpericyte interactions strongly induce TIMP-3 expression by pericytes, whereas ECs produce TIMP-2 in ECāpericyte cocultures. Using small interfering RNA technology, the suppression of EC TIMP-2 and pericyte TIMP-3 expression leads to capillary tube regression in these cocultures in a matrix metalloproteinase-1 (MMP-1)ā, MMP-10ā, and ADAM-15 (a disintegrin and metalloproteinase-15)ādependent manner. Furthermore, we show that EC tube morphogenesis (lumen formation and invasion) is primarily controlled by the TIMP-2 and -3 target membrane type (MT) 1 MMP. Additional targets of these inhibitors include MT2-MMP and ADAM-15, which also regulate EC invasion. Mutagenesis experiments reveal that TIMP-3 requires its proteinase inhibitory function to induce tube stabilization. Overall, these data reveal a novel role for both TIMP-2 and -3 in the pericyte-induced stabilization of newly formed vascular networks that are predisposed to undergo regression and reveal specific molecular targets of the inhibitors regulating these events
AI is a viable alternative to high throughput screening: a 318-target study
: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNetĀ® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNetĀ® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery
Intrinsic Dynamics Prime Calmodulin for Peptide Binding: Characterizing Lowly Populated States by Paramagnetic Relaxation Enhancement
Structural studies of integrin activation
Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that link the extracellular matrix to the actin cytoskeleton. Uniquely, these adhesion receptors mediate inside-out signal transduction, whereby extracellular adhesion is activated from within the cell by talin, a large cytoskeletal protein that binds to the cytoplasmic tail of the Ī² integrin subunit via its PTB-like F3 domain. Features of the interface between talin1 and small Ī²3 fragments only have been described previously. Through NMR studies of full-length integrin Ī² tails, we have found that Ī² tails differ widely in their interactions with different talin isoforms. The muscle-specific Ī²1D/talin2 complex exhibited particularly high affinity, leading to the X-ray crystal structure of the Ī²1D tail/talin2 F2-F3 complex. Further NMR and biological experiments demonstrated that integrin activation is induced by a concerted series of interactions between the talin F3 domain and the Ī² tail and between the talin F2 domain and the cell membrane. Additional studies revealed the structural determinants of tight talin2/Ī²1D binding and the basis of more general differences between Ī²1 and Ī²3 talin binding. NMR studies were also performed on tyrosine-phosphorylated integrin tails binding to the PTB domains of talin1 and Dok1, an inhibitor of integrin activation; these revealed that phosphorylation can inhibit integrin activation by increasing the affinity of the Ī² tail for talin competitors. Key residues governing this switch were identified, and proteins were engineered with reversed affinities, offering potentially useful biological tools. Taken together, these results reveal the remarkable complexity of structural features that enable talin and its competitors to mediate this important form of transmembrane signalling.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
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The Global Challenge to Prevent Breast Cancer: Surfacing New Ideas to Accelerate Prevention Research
Despite increases in screening and advances in treatment, breast cancer continues to be the most common cancer and cause of cancer deaths among women worldwide, and breast cancer rates have remained steady for decades. A new focus on population-level primary prevention is needed to tackle this disease at the most fundamental level. Unfortunately, only a small fraction of breast cancer research funds currently go to prevention. The California Breast Cancer Research Program (CBCRP) seeks to change this. In order to accelerate breast cancer primary prevention efforts, in 2018, CBCRP launched the Global Challenge to Prevent Breast Cancer, a prize competition to foster and disseminate new and innovative prevention research ideas. This Special Issue highlights the results of the Global Challenge and other CBCRP primary prevention efforts
The Global Challenge to Prevent Breast Cancer: Surfacing New Ideas to Accelerate Prevention Research
Despite increases in screening and advances in treatment, breast cancer continues to be the most common cancer and cause of cancer deaths among women worldwide, and breast cancer rates have remained steady for decades. A new focus on population-level primary prevention is needed to tackle this disease at the most fundamental level. Unfortunately, only a small fraction of breast cancer research funds currently go to prevention. The California Breast Cancer Research Program (CBCRP) seeks to change this. In order to accelerate breast cancer primary prevention efforts, in 2018, CBCRP launched the Global Challenge to Prevent Breast Cancer, a prize competition to foster and disseminate new and innovative prevention research ideas. This Special Issue highlights the results of the Global Challenge and other CBCRP primary prevention efforts