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Abstract p21WAF1/CIP1 (p21) plays critical roles in cell‐cycle regulation and DNA repair and is transcriptionally regulated through p53‐dependent or ‐independent pathways. Bioinformatic analysis predicated one stress‐response element (STRE) implicated in single nucleotide polymorphism (SNP) rs2395655 of the p21 promoter. Here, we investigated the transcriptional regulatory function of rs2395655 variant genotype and analyzed its associations with the p21 expression and clinical outcomes in esophageal squamous cell carcinoma (ESCC) patients. Luciferase assay results showed significantly increased transcriptional activity of the rs2395655 G allele‐containing p21 promoter compared with rs2395655 A allele‐containing counterpart, especially in ESCC cells with ectopic LEDGF/p75 expression. Furthermore electrophoretic mobility shift assay using the rs2395655 G or A allele‐containing probe and chromatin immunoprecipitation assay with specific anti‐LEDGF/p75 antibody indicated the potential binding activity of LEDGF/p75 with the STRE element implicated in rs2395655 G allele of the p21 promoter. Subsequent specific RNA interference‐mediated depletion or ectopic expression of LEDGF/p75 caused obviously down‐ or up‐regulated expression of p21 mRNA in ESCC cells harboring rs2395655 GG genotype but not cells with rs2395655 AA genotype. Furthermore, rs2395655 GG genotype carriers showed significantly elevated p21 protein expression and conferred survival advantage in both univariate and multivariate analyses in total 218 ESCC patients. Our findings suggest that LEDGF/p75 regulates the p21 expression in ESCC cells through interacting with STRE element implicated in polymorphism rs2395655 and the elevated p21 protein expression and rs2395655 GG genotype may serve as positive prognostic factors for ESCC patients.

p21WAF1/CIP1 (p21) plays critical roles in cell‐cycle regulation and DNA repair and is transcriptionally regulated through p53‐dependent or ‐independent pathways. Bioinformatic analysis predicated one stress‐response element (STRE) implicated in single nucleotide polymorphism (SNP) rs2395655 of the p21 promoter. Here, we investigated the transcriptional regulatory function of rs2395655 variant genotype and analyzed its associations with the p21 expression and clinical outcomes in esophageal squamous cell carcinoma (ESCC) patients. Luciferase assay results showed significantly increased transcriptional activity of the rs2395655 G allele‐containing p21 promoter compared with rs2395655 A allele‐containing counterpart, especially in ESCC cells with ectopic LEDGF/p75 expression. Furthermore electrophoretic mobility shift assay using the rs2395655 G or A allele‐containing probe and chromatin immunoprecipitation assay with specific anti‐LEDGF/p75 antibody indicated the potential binding activity of LEDGF/p75 with the STRE element implicated in rs2395655 G allele of the p21 promoter. Subsequent specific RNA interference‐mediated depletion or ectopic expression of LEDGF/p75 caused obviously down‐ or up‐regulated expression of p21 mRNA in ESCC cells harboring rs2395655 GG genotype but not cells with rs2395655 AA genotype. Furthermore, rs2395655 GG genotype carriers showed significantly elevated p21 protein expression and conferred survival advantage in both univariate and multivariate analyses in total 218 ESCC patients. Our findings suggest that LEDGF/p75 regulates the p21 expression in ESCC cells through interacting with STRE element implicated in polymorphism rs2395655 and the elevated p21 protein expression and rs2395655GG genotype may serve as positive prognostic factors for ESCC patients.We demonstrated for the first time that LEDGF/p75 binds to STRE element implicated in polymorphism rs2395655 and regulates the p21 gene expression in ESCC cells and tissues. Our results suggested that the elevated p21 protein expression and rs2395655 GG variant may serve as positive prognostic factors for ESCC patients. [ABSTRACT FROM AUTHOR]

Lens epithelium‐derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV‐1 integrase in human cells. The crystal structure of the HIV integrase‐binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting‐drop vapour diffusion. X‐ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain‐swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant. [ABSTRACT FROM AUTHOR]

Integrase-binding domain (IBD) is a critical region on the lens epithelium-derived growth factor (LEDGF/p75) protein that specifically interacts with human immunodeficiency virus type 1 integrase (HIV-1 IN). In this study, we expressed and purified the IBD, and utilized in endonucleolytic assay and strand transfer assay to investigate whether IBD improve enzymatic activities of HIV-1 IN. Our results showed that IBD along with HIV-1 IN increased the strand transfer activity but not the endonucleolytic activity. Furthermore, our results showed that IBD induced efficient integration of the donor DNA into the target DNA to create half-site integration and full-site integration using plasmid as target DNA. Therefore, it is suggested that LEDGF-IBD itself might be able to increase viral infectivity by enhancing the strand transfer activities of HIV-1 IN and accelerating integration of viral cDNA into cellular chromosomal DNA. [ABSTRACT FROM AUTHOR]

Abstract: Lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional coactivators that connect sequence-specific activators to the basal transcription machinery. We have found that these proteins are posttranslationally modified by SUMO (small ubiquitin-like modifier)-1 and SUMO-3. Three SUMOylation sites, K75, K250, and K254, were mapped on the shared N-terminal region of these molecules, while a fourth site, K364, was identified in the C-terminal part exclusive of LEDGF/p75. The N-terminal SUMO targets are located in evolutionarily conserved charge-rich regions that lack resemblance to the described consensus SUMOylation motif, whereas the C-terminal SUMO target is solvent exposed and situated in a typical consensus motif. SUMOylation did not affect the cellular localization of LEDGF proteins and was not necessary for their chromatin-binding ability, nor did it affect this activity. However, lysine to arginine mutations of the identified SUMO acceptor sites drastically inhibited LEDGF SUMOylation, extended the half-life of LEDGF/p75, and significantly increased its transcriptional activity on the heat shock protein 27 promoter, indicating a negative effect of SUMOylation on the transcriptional activity of LEDGF/p75. Considering that SUMOylation is known to negatively affect the transcriptional activity of all transcription factors known to transactivate heat shock protein 27 expression, these findings support the paradigm establishing SUMOylation as a global neutralizer of cellular processes upregulated upon cellular stress. [Copyright &y& Elsevier]

TNF-α induces oxidative stress by generating reactive oxygen species (ROS). This molecule elevates the expression of γ-glutamylcysteine synthetase heavy subunit (γ-GCS-HS). Lens epithelium-derived growth factor (LEDGF)/p75. a transcriptional protein, is inducible by oxidative stress and protects ceils from various stresses by upregulating stress-responsive genes. This paper presents evidence that TNF-α elevates the expression of LEDGF and that LEDGF is one of the transactivators of γ-GCS-HS gene. An analysis of the γ-GCS-HS promoter sequence (-819 to +518 nt) revealed the presence of putative sites for LEDGF binding. Gel mobility assay confirmed the binding of LEDGF to the heat shock element (nGAAn) and the stress response element (A/TGGGGA/T) present in γ-GCS-HS promoter. Transactivation experiments showed activation of γ-GCS-HS promoter in cells overexpressing LEDGF or treated with a sublethal dose of TNF-α (20 ng/ml). Downregulation of γ-GCS-HS promoter activity in cells transfected with LEDGF small interfering RNA validated the finding. Notably, cells treated with TNF-α (20 ng/ml) for 24 h had an increased abundance of LEDGF and γ-GCS-HS mRNA and protein. In contrast, cells treated with TNF-α for longer periods or with higher concentrations of TNF-α showed reduced expression of LEDGF and γ-GCS-HS and increased cellular death with higher ROS levels. Cells overexpressing LEDGF revealed elevated GSH levels (10–15%), a condition that may potentially eliminate the insult to cells induced by TNF-α. Thus TNF-α regulation of LEDGF may be physiologically important, as elevated expression of LEDGF increases the expression of endogenous γ-GCS-HS gene, the catalytic subunit of the regulating enzyme in GSH biosynthesis that may constitute a protective mechanism in limiting oxidative stress induced by inflammatory cytokines. [ABSTRACT FROM AUTHOR]

Lens epithelium derived growth factor (LEDGF), a nuclear protein, plays a role in regulating the transcription of stress-associated genes such as heat shock proteins by binding to consensus core DNA sequences nAGGn or nGAAn or their repeats, and in doing so helps to provide cyto-protection. However, additional information is required to identify the specific structural features of LEDGF involved in gene transcription. Here we have investigated the functional domains activating and repressing DNA-binding modules, by using a DNA binding assay and trans-activation experiments performed by analyzing proteins prepared from deletion constructs. The results disclosed the DNA-binding domain of N-terminal LEDGF mapped between amino acid residues 5 and 62, a 58 amino acid residue stretch PWWP domain which binds to stress response elements (STRE; A/TGGGGA/T). C-terminal LEDGF contains activation domains, an extensive loop-region (aa 418–530) with two helix-turn-helix (HTH)-like domains, and binds to a heat shock element (HSE; nGAAn). A trans-activation assay using Hsp27 promoter revealed that both HTH domains contribute in a cooperative manner to the trans-activation potential of LEDGF. Interestingly, removal of N-terminal LEDGF (aa 1–187) significantly enhances the gene activation potential of C-terminal LEDGF (aa 199–530); thus the N-terminal domain (aa 5–62), exhibits auto-transcriptional repression activity. It appears that this domain is involved in stabilizing the LEDGF–DNA binding complex. Collectively, our results demonstrate that LEDGF contains three DNA-binding domains, which regulate gene expression depending on cellular microenvironment and thus modify the physiology of cells to maintain cellular homeostasis. [Copyright &y& Elsevier]

Abstract: Lens epithelium-derived growth factor (LEDGF/p75) is a novel transcription co-activator that is critically involved in lens epithelial cell gene regulation and stress responses. Recent evidence indicates that LEDGF/p75 may play an important role in lens epithelial to fibre cell terminal differentiation. Since the lens and the brain are both ectodermally derived organs generated from epithelioid progenitor cells, we hypothesize that LEDGF/p75 is expressed and subserving similar functions in both organs. To investigate this hypothesis, we studied LEDGF/p75 expression and localization in the human brain. We detected LEDGF/p75-specific RT-PCR reaction products in both fetal and adult human brain. LEDGF/p75 mRNA expression in the brain exhibited differential developmental and regional specificity. LEDGF/p75 transcript was markedly elevated in fetal as compared to adult brain. In the adult brain, LEDGF/p75 mRNA expression was substantial in the subventricular zone (SVZ), scant in hippocampus, and undetectable elsewhere. To study LEDGF/p75 protein expression and localization, we developed and purified a new anti-LEDGF/p75 polyclonal antibody directed against a unique C-terminal region of LEDGF/p75. Western blot analysis of fetal and adult human brain revealed a ∼75kDa protein that demonstrated developmental and regional specificity similar to that detected by RT-PCR analysis. LEDGF/p75 protein expression was high in fetal brain and in the adult SVZ. Immunohistochemical studies of human fetal brain showed prominent LEDGF/p75-immunoreactive cells in the germinal neuroepithelium and cortical plate regions. Analysis of adult and aged human brain revealed LEDGF/p75-immunoreactive cell enrichment in the SVZ adjacent to the ventral region of the lateral ventricle at the level of the anterior commissure, a region implicated in adult neurogenesis. We utilised a primary mixed cortical cell culture system to identify LEDGF/p75 in neurons, but not astrocytes. Neuronal LEDGF/p75 exhibited a predominantly perinuclear distribution pattern. These data demonstrate that LEDGF/p75 is expressed in discrete regions and cell types within the fetal and adult human brain. Moreover, the developmental and regional expression patterns of LEDGF/p75 suggest that this transcriptional co-activator may be involved in neuroepithelial stem cell differentiation and neurogenesis. [Copyright &y& Elsevier]

Retinoic acid (RA) is required for the normal growth and maintenance of many cell types, including lens epithelial cells (LECs). Alcohol (ADH) and aldehyde (ALDH) dehydrogenases are implicated in cellular detoxification and conversion of vitamin A to RA. Lens epithelium-derived growth factor (LEDGF) provides cellular protection against stress by transactivating stress-associated genes. Here we show evidence that LEDGF binds and transactivates heat shock (nGAAn) and stress response (A/TGGGGAJT) elements in the promoters of ADH1, ADH4, and retinaldehyde 2 (RALDH2) genes. Electrophoretic mobility and supershift assays disclosed specific binding of LEDGF to nGAAn and A/TGGGGA/T elements in these gene promoters. Transfection experiments in LECs with promoters linked to a chloramphenicol acetyltransferase (CAT) reporter gene along with LEDGF cDNA revealed higher CAT activity. RTPCR results confirmed that LECs overexpressing LEDGF contained increased levels of ADH1, ADH4, and RALDH2 mRNA. Notably, LECs displayed higher LEDGF mRNA and protein expression during ethanol stress. Cells overexpressing LEDGF typically exhibited elevated RA levels and survived well during ethanol stress. The present findings indicate that LEDGF is one of the transcriptional activators of these genes that facilitates cellular protection against ethanol stress and plays a role in RA production. [ABSTRACT FROM AUTHOR]

The purpose of this study is to investigate possible neuroprotective effects of lens epithelium-derived growth factor (LEDGF) against cell death induced by N-methyl-d-aspartate (NMDA) in the rat retina. LEDGF and/or NMDA were intravitreally injected into rat eyes. NMDA-induced retinal death and protective effects of LEDGF were evaluated by morphometric analysis, cell numbers in the ganglion cell layer (GCL) and the thickness of the inner plexiform layer (IPL). Retrograde labeling with a fluorescent tracer (Fluoro-Gold) was applied for counting retinal ganglion cells (RGCs) that survived after NMDA injection. Terminal deoxyribonucleotidyl transferase (TdT)-mediated fluroscein-16-dUTP nick end-labeling (TUNEL) staining was used to evaluate of retinal cell death. Morphometric analysis and retrograde labeling analysis showed that retinal damage induced by NMDA was protected significantly by LEDGF. TUNEL assay revealed that pretreatment with LEDGF prevents NMDA-induced apoptosis. Retinal damage (ganglion and amacrine cells) induced by NMDA was protected by an intravitreal injection of LEDGF. [Copyright &y& Elsevier]

Indirect immunofluorescence (IIF) is the most prevalent screening antinuclear antibody test for systemic autoimmune rheumatic disease (SARD). Certain IIF patterns have known antibody and disease associations, but the dense fine speckled (ANA-DFS) pattern has no confirmed clinical associations. Our objective was to determine the prevalence of SARD among a group of ANA-DFS positive individuals and to identify final diagnoses among non-SARD individuals in order to determine possible clinical associations with the ANA-DFS pattern. A retrospective study of 425 patients from a university health care system with a positive ANA-DFS pattern consecutively between August 2017 and September 2018. Sera samples underwent ANA testing by IIF on HEp-2 cell substrates (Euroimmun, Germany). Clinical information was retrieved from electronic health records and stored in a de-identified database. The prevalence of SARD was 24%. Undetermined diagnosis (17%), skin disorders (12.1%), and fibromyalgia/chronic pain syndrome/chronic fatigue syndrome (11.8%) were the most common non-SARD diagnoses. Taking into account past medical history, the most common non-SARD were atopic disorders (21.2%), fibromyalgia/chronic pain syndrome/chronic fatigue syndrome (17.6%), and skin disorders (16.7%). The ANA-DFS pattern may be indicative of an underlying antigen-antibody interaction that plays a role in either the initiation or propagation of immunologic reactions. DFS70/LEDGF is a transcription factor involved in cell survival and stress protection, and autoantibodies may inhibit its function. It is likely that there are other antibodies producing the ANA-DFS pattern besides anti-DFS70/LEDGF, and more research is necessary to identify additional antibody specificities. The ANA-DFS pattern may be an indicator of a proinflammatory microenvironment given the high frequency of symptomatic patients and disease processes with an immunologic basis (including SARD). • ANA-DFS pattern may be an indicator of a proinflammatory microenvironment. • ANA-DFS pattern cannot exclude the presence of systemic autoimmune rheumatic disease. • Possible associations with ANA-DFS are skin disorders, atopic disorders, fibromyalgia. • The development of autoantibodies to LEDGF may inhibit its pro-survival function. • Other antibodies are likely producing the ANA-DFS pattern besides anti-DFS70/LEDGF. [ABSTRACT FROM AUTHOR]

The disruption of crucial interactions between HIV-1 Integrase and cellular cofactor LEDGF/p75 represents an emerging approach for the design and development of new antiretroviral agents. In this study we report the successful application of a structure-based virtual screening strategy for the discovery of natural hit structures able to inhibit Integrase-LEDGF/p75 interaction. The application of sequential filters (drug-likeness, 3D-pharmacophore mapping, docking, molecular dynamics simulations) yielded a hit list of compounds, out of which 9 were tested in the in vitro AlphaScreen assays and 8 exhibited a detectable inhibition of the interaction between the two proteins. The best inhibitors belong to different chemical classes and could be represent a good starting point for further optimization and structure-activity relationship studies., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

For vision-threatening retinitis pigmentosa and dry age-related macular degeneration, there are no United States Food and Drug Administration (FDA)-approved treatments. We identified, biosynthesized, purified, and characterized lens epithelium-derived growth factor fragment (LEDGF1-326) as a novel protein therapeutic. LEDGF1-326 was produced at about 20 mg/liter of culture when expressed in the Escherichia coli system, with about 95% purity and aggregate-free homogeneous population with a mean hydrodynamic diameter of 9 ± 1 nm. The free energy of unfolding of LEDGF1-326 was 3.3 ± 0.5 kcal mol(-1), and melting temperature was 44.8 ± 0.2 °C. LEDGF1-326 increased human retinal pigment epithelial cell viability from 48.3 ± 5.6 to 119.3 ± 21.1% in the presence of P23H mutant rhodopsin-mediated aggregation stress. LEDGF1-326 also increased retinal pigment epithelial cell FluoSphere uptake to 140 ± 10%. Eight weeks after single intravitreal injection in Royal College of Surgeons (RCS) rats, LEDGF1-326 increased the b-wave amplitude significantly from 9.4 ± 4.6 to 57.6 ± 8.8 μV for scotopic electroretinogram and from 10.9 ± 5.6 to 45.8 ± 15.2 μV for photopic electroretinogram. LEDGF1-326 significantly increased the retinal outer nuclear layer thickness from 6.34 ± 1.6 to 11.7 ± 0.7 μm. LEDGF1-326 is a potential new therapeutic agent for treating retinal degenerative diseases.