Dual-site recognition of different extracellular matrix components by anti-angiogenic/neurotrophic serpin,PEDF

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) superfamily, possesses anti-angiogenic and neurotrophic activities. PEDF has been reported to bind to extracellular matrix (ECM) components such as collagens and glycosaminoglycans (GAGs). In this study, to determine the binding sites for collagens and GAGs, we analyzed the interaction of recombinant mouse PEDF (rPEDF) with collagen I and heparin. By utilizing residue-specific chemical modification and site-directed mutagenesis techniques, we revealed that the acidic amino acid residues on PEDF (Asp(255), Asp(257), and Asp(299)) are critical to collagen binding, and three clustered basic amino acid residues (Arg(145), Lys(146), and Arg(148)) are necessary for heparin binding. Mapping of these residues on the crystal structure of human PEDF (Simonovic, M., Gettins, P. G. W., and Volz, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11131-11135) demonstrated that the collagen-binding site is oriented toward the opposite side of the highly basic surface where the heparin-binding site is localized. These results indicate that PEDF possesses dual binding sites for different ECM components, and this unique localization of ECM-binding sites implies that the binding to ECM components could regulate PEDF activities.     

Pigment epithelium-derived factor (PEDF), a serpin with potent anti-angiogenic and neurite outgrowth-promoting properties

Pigment epithelium-derived factor is a member of the serpin superfamily of proteins, but one that lacks inhibitory properties against either serine or cysteine proteinases. Nevertheless it possesses a number of physiological properties that make it a potentially important protein in regulation of angiogenesis, in neuronal cell survival and in protection of neurons from neurotoxic agents. It is also a protein that is highly up-regulated in the G0 phase of early-passage cells compared with rapidly proliferating cells or senescent cells, and so is also linked to both the cell cycle and cell senescence. The determination of a high resolution X-ray crystal structure of native PEDF provides insight into regions of the protein that may be involved in one or more of these functions.     

Proliferative diabetic retinopathy is associated with a low level of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor. a pilot study.

Retinopathy is the most common microvascular diabetes complication and represents a major threat to the eyesight. The aim of this study was to address the role of pro- and anti-angiogenic molecules in diabetic retinopathy in the aqueous humor of the eye. Aqueous humor was collected at cataract surgery from 19 diabetic patients and from 13 age- and sex-matched normoglycemic controls. Levels of pro-angiogenic vascular endothelial growth factor (VEGF) and angiogenic inhibitor pigment epithelium-derived factor (PEDF) were determined. Angiogenic activity of the aqueous humor was quantified by measuring its effect on the migration of capillary endothelial cells. In the aqueous fluid, VEGF levels were increased in diabetics (mean values: 501 vs. 367 pg/ml; p = 0.05), compared to controls. PEDF was found to be decreased in diabetics (mean values: 2080 vs. 5780 ng/ml; p = 0.04) compared to controls. In seven diabetic patients with proliferative retinopathy, the most profound finding was a significant decrease of the PEDF level (mean value: 237 ng/ml), whereas VEGF levels were comparable to diabetic patients without proliferation (mean value: 3153; p = 0.003). Angiogenic activity in samples of patients from the control group was generally inhibitory due to PEDF, and inhibition was blocked by neutralizing antibodies to PEDF. Likewise, in diabetics without proliferation, angiogenic activity was also blocked by antibodies to PEDF. We will demonstrate here that the level of the natural ocular anti-angiogenic agent PEDF is inversely associated with proliferative retinopathy. PEDF is an important negative regulator of angiogenic activity of aqueous humor. Our data may have implications for the development of novel regimens for diabetic retinopathy.     

VE-cadherin links tRNA synthetase cytokine to anti-angiogenic function

A natural fragment of an enzyme that catalyzes the first step of protein synthesis-human tryptophanyl-tRNA synthetase (T2-TrpRS) has potent anti-angiogenic activity. A cellular receptor through which T2-TrpRS exerts its anti-angiogenic activity has not previously been identified. Here T2-TrpRS was shown to bind at intercellular junctions of endothelial cells (ECs). Using genetic knock-outs, binding was established to depend on VE-cadherin, a calcium-dependent adhesion molecule, which is selectively expressed in ECs, concentrated at adherens junctions, and is essential for normal vascular development. In contrast, T2-TrpRS binding to EC junctions was not dependent on platelet endothelial cell adhesion molecule type-1, another adhesion molecule found at EC junctions. Pull-down assays confirmed direct complex formation between T2-TrpRS and VE-cadherin. Binding of T2-TrpRS inhibited VEGF-induced ERK activation and EC migration. Thus, a VE-cadherin-dependent pathway is proposed to link T2-TrpRS to inhibition of new blood vessel formation.     

The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

Cardiac diseases such as myocardial infarction and heart failure are among the leading causes of death in western societies. Therapeutic angiogenesis has been suggested as a concept to combat these diseases. The biology of angiogenic factors expressed in the heart such as vascular endothelial growth factor (VEGF) is well studied, whereas data on anti-angiogenic mediators in the heart are scarce. Here we study the expression of the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the human heart and in human cardiac cells. PEDF expression could be detected in human cardiac tissue on the protein and mRNA levels. PEDF mRNA levels were significantly lower in explanted human ischemic hearts as compared to healthy hearts. Our in vitro experiments showed that human adult cardiac myocytes and fibroblasts constitutively secrete PEDF. In addition to anoxic conditions, cobalt chloride, 2,2'dipyridyl and dimethoxally glycine, which stabilize hypoxia inducible factor-α decreased PEDF expression. Furthermore we show that PEDF inhibits VEGF-induced sprouting. We have identified PEDF in healthy and ischemic human hearts and we show that PEDF expression is down-regulated by low oxygen levels. Therefore, we suggest a role for PEDF in the regulation of angiogenesis in the heart and propose PEDF as a possible therapeutic target in heart disease.     

Shugoshins function as a guardian for chromosomal stability in nuclear division

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.     

Shugoshins function as a guardian for chromosomal stability in nuclear division

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.     

Centromere cohesion： regulating the guardian

Mitosis, a process in which duplicated chromosomes are segregated into two daughter cells, is the most spectacular event in cell cycle. In mitosis, cells undergo drastic rearrangement of cytoskeleton, lipid and chromosomes under amazingly strict temporal and spatial control so that genetic information is faithfully transmitted to daughter cells.[第一段]     

Cohesin: A guardian of genome integrity

Ability to reproduce is one of the hallmark features of all life forms by which new organisms are produced from their progenitors. During this process each cell duplicates its genome and passes a copy of its genome to the daughter cells along with the cellular matrix. Unlike bacteria, in eukaryotes there is a definite time gap between when the genome is duplicated and when it is physically separated. Therefore, for precise halving of the duplicated genome into two, it is required that each pair of duplicated chromosomes, termed sister chromatids, should be paired together in a binary fashion from the moment they are generated. This pairing function between the duplicated genome is primarily provided by a multimeric protein complex, called cohesin. Thus, genome integrity largely depends on cohesin as it ensures faithful chromosome segregation by holding the sister chromatids glued together from S phase to anaphase. In this review, we have discussed the life cycle of cohesin during both mitotic and meiotic cell divisions including the structure and architecture of cohesin complex, relevance of cohesin associated proteins, mechanism of cohesin loading onto the chromatin, cohesion establishment and the mechanism of cohesin disassembly during anaphase to separate the sister chromatids. We have also focused on the role of posttranslational modifications in cohesin biology. For better understanding of the complexity of the cohesin regulatory network to the readers, we have presented an interactome profiling of cohesin core subunits in budding yeast during mitosis and meiosis.     

Shugoshin: a centromeric guardian senses tension

To ensure accurate chromosome segregation during mitosis, the spindle checkpoint monitors chromosome alignment on the mitotic spindle. Indjeian and colleagues have investigated the precise role of the shugoshin 1 protein (Sgo1p) in this process in budding yeast. The Sgo proteins were originally identified as highly conserved proteins that protect cohesion at centromeres during the first meiotic division. Together with other recent findings, the study highlighted here has identified Sgo1 as a component that informs the mitotic spindle checkpoint when spindle tension is perturbed. This discovery has provided a molecular link between sister chromatid cohesion and tension-sensing at the kinetochore-microtubule interface.     

Shugoshin: guardian spirit at the centromere

A recently emerging protein family, shugoshin, plays a crucial role in the centromeric protection of cohesin, which is responsible for sister chromatid cohesion. This is especially important at the first meiotic division, where cohesin is cleaved by separase only along chromosome arms while the centromeric cohesin must be preserved. In vertebrate cells, arm cohesion is largely lost during prophase and prometaphase in order to facilitate sister chromatid resolution, whereas centromeric cohesion is preserved until the bipolar attachment of sister chromatids is established. Vertebrate shugoshin plays an essential role in protecting centromeric cohesin from prophase dissociation. In yeast, shugoshin also has a crucial role in sensing the loss of tension at kinetochores and in generating the spindle checkpoint signal.     

Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.     

UHRFI:a novel metabolic guardian restricting AMPK activity

AMP-activated protein kinase (AMPK) serves as a central guardian maintaining energy homeostasis under nutrient stress and/or energy crisis.The study by Xu et al...     

RecQ helicases: guardian angels of the DNA replication fork

Since the original observations made in James German’s Laboratory that Bloom’s syndrome cells lacking BLM exhibit a decreased rate of both DNA chain elongation and maturation of replication intermediates, a large body of evidence has supported the idea that BLM, and other members of the RecQ helicase family to which BLM belongs, play important roles in DNA replication. More recent evidence indicates roles for RecQ helicases in what can broadly be defined as replication fork ‘repair’ processes when, for example, forks encounter lesions or adducts in the template, or when forks stall due to lack of nucleotide precursors. More specifically, several roles in repair of damaged forks via homologous recombination pathways have been proposed. RecQ helicases are generally only recruited to sites of DNA replication following fork stalling or disruption, and they do so in a checkpoint-dependent manner. There, in addition to repair functions, they aid the stabilisation of stalled replication complexes and seem to contribute to the generation and/or transduction of signals that enforce S-phase checkpoints. RecQ helicases also interact physically and functionally with several key players in DNA replication, including RPA, PCNA, FEN1 and DNA polymerase δ. In this paper, we review the evidence that RecQ helicases contribute to the impressively high level of fidelity with which genome duplication is effected.     

H2AX: the histone guardian of the genome

At close hand to one's genomic material are the histones that make up the nucleosome. Standing guard, one variant stays hidden doubling as one of the core histones. But, thanks to its prime positioning, a variation in the tail of H2AX enables rapid modification of the histone code in response to DNA damage. A role for H2AX phosphorylation has been demonstrated in DNA repair, cell cycle checkpoints, regulated gene recombination events, and tumor suppression. In this review, we summarize what we have learned about this marker of DNA breaks, and highlight some of the questions that remain to be elucidated about the physiological role of H2AX. We also suggest a model in which chromatin restructuring mediated by H2AX phosphorylation serves to concentrate DNA repair/signaling factors and/or tether DNA ends together, which could explain the pleotropic phenotypes observed in its absence.     

Bone marrow microenvironment: The guardian of leukemia stem cells

Bone marrow microenvironment(BMM) is the main sanctuary of leukemic stem cells(LSCs) and protects these cells against conventional therapies. However, it may open up an opportunity to target LSCs by breaking the close connection between LSCs and the BMM. The elimination of LSCs is of high importance, since they follow cancer stem cell theory as a part of this population. Based on cancer stem cell theory, a cell with stem cell-like features stands at the apex of the hierarchy and produces a heterogeneous population and governs the disease.Secretion of cytokines, chemokines, and extracellular vesicles, whether through autocrine or paracrine mechanisms by activation of downstream signaling pathways in LSCs, favors their persistence and makes the BMM less hospitable for normal stem cells. While all details about the interactions of the BMM and LSCs remain to be elucidated, some clinical trials have been designed to limit these reciprocal interactions to cure leukemia more effectively. In this review, we focus on chronic myeloid leukemia and acute myeloid leukemia LSCs and their milieu in the bone marrow, how to segregate them from the normal compartment, and finally the possible ways to eliminate these cells.