Relation between the spatial structure and antigenic activity of trophoblast-specific beta1-glycoprotein

Temperature- and pH-dependence of spatial structure of a native trophoblast-specific beta-glycoprotein (TSG), its desialated and deglycosylated derivatives, as well as of a fragment obtained by partial acid hydrolysis on the temperature and pH variations has been studied using CD and UV spectroscopy. Within the range 45-50 degrees C a conformational transition of the protein moiety of TSG, leading to partially reversible alterations in tertiary and secondary structures of this glycoprotein after cooling the solution to 20 degrees C has been found out. The results of spectral studies of the spatial structure of the TSG protein component have been compared with the data on antigen activity of native, temperature- and pH-denaturated, desialated, and deglycosilated TSG. It has been concluded that the protein moiety of TSG consists mainly of beta-structures; the greater part of antigen determinants of TSG is topographic and belongs to the protein component of TSG, and only 15% of antigen determinants are not connected with TSG's spatial structure.     

Characteristics of the carbohydrate component of trophoblastic beta1-glycoprotein

By use of the methylation method, trophoblast-specific beta 1-glycoprotein (TSG, SP-1) has been shown to contain N-glycosidically linked oligosaccharide chains of the N-acetyl lactosamine type. Methylation analysis of TSG fractions which have various affinity to concanavalin A indicates that the diantennary carbohydrate chains prevail in the glycoprotein molecule.     

Physico-chemical properties of trophoblast-specific beta 1-globulin

It has been found that the trophoblast-specific beta 1-globulin [TSG) contains one site of binding to Con A and phytohemagglutinin. The interaction with phytohemagglutinin should be taken into consideration, when studying the immunosuppressive activity of the protein. Additional fractions of the protein with fast anodic electrophoretic mobility and lower molecular weights have been detected after treatment of the trophoblast-specific beta 1-globulin with diethylene triaminopentaacetate. This is indicative of a subunit structure of the glycoprotein molecules.     

Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in chondrocytes in vitro and in vivo

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.     

Localization of antigenic determinants on a molecule of trophoblast-specific beta 1-glycoprotein

Spatial localization of antigenic determinants of trophoblast-specific beta I-glycoprotein (TSG) has been elucidated using chemical modifications of the sugar and protein moieties of the molecule. Various deglycosylation procedures of TSG afforded fragments slightly soluble even in the presence of powerful detergents. Treatment of TSG with boric acid and its salts, accompanied with a conformational change of the sugar moiety, failed to alter conformation of the protein portion as evidenced by CD spectral data. This modification was found to increase the antigenic activity of TSG only scarcely. Modification of tryptophane or tyrosine residues of TSG changed spatial structure of the protein portion to can be a considerable loss of the TSG antigenic activity. The data obtained led to the conclusion that antigenic determinants of TSG are localized at the protein portion of the molecule and are topographic. A tryptophane residue is an indispensable constituent of the antigenic determinants.     

Twisted gastrulation loss-of-function analyses support its role as a BMP inhibitor during early Xenopus embryogenesis

BMP signals play important roles in the regulation of diverse events in development and in the adult. In amniotes, like the amphibian Xenopus laevis, BMPs promote ventral specification, while chordin and other BMP inhibitors expressed dorsally in the Spemann's organizer play roles in establishment and/or maintenance of this region as dorsal endomesoderm. The activities of chordin are in turn regulated by the secreted proteolytic enzymes BMP1 and Xolloid. Recently, we and others have identified the protein twisted gastrulation (TSG) as a soluble BMP modulator that functions by modifying chordin activity. Overexpression and genetic analyses in Drosophila, Xenopus and zebrafish together with in vitro biochemical studies suggest that TSG might act as a BMP antagonist; but there is also evidence that TSG may promote BMP signaling. Here we report examination of the in vivo function of TSG in early Xenopus development using a loss-of-function approach. We show that reducing TSG expression using antisense TSG morpholino oligonucleotides (MOs) results in moderate head defects. These defects can be rescued both by a TSG that cannot be inhibited by the MO, and by the BMP antagonists chordin and noggin. Furthermore, while neither the onset of gastrulation nor the expression of marker genes are affected in early gastrulae, dorsal marker gene expression is reduced at the expense of expanded ventral marker gene expression beginning at mid to late gastrula stage. TSG-MO and Chd-MOs also cooperate to strongly repress head formation. Finally, we note that the loss of TSG function results in a shift in tissue responsiveness to the BMP inhibitory function of chordin in both animal caps and the ventral marginal zone, a result that implies that the activity of TSG may be required for chordin to efficiently inhibit BMPs in these developmental contexts. These data, taken together with the biochemistry and overexpression studies, argue that TSG plays an important role in regulating the potency of chordin's BMP inhibitory activity and TSG and chordin act together to regulate the extent of dorsoanterior development of early frog embryos.     

The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.     

Ubiquitin recognition by the human TSG101 protein

The UEV domain of the TSG101 protein functions in both HIV-1 budding and the vacuolar protein sorting (VPS) pathway, where it binds ubiquitylated proteins as they are sorted into vesicles that bud into late endosomal compartments called multivesicular bodies (MVBs). TSG101 UEV-ubiquitin interactions are therefore important for delivery of both substrates and hydrolytic enzymes to lysosomes, which receive proteins via fusion with MVBs. Here, we report the crystal structure of the TSG101 UEV domain in complex with ubiquitin at 2.0 A resolution. TSG101 UEV contacts the Ile44 surface and an adjacent loop of ubiquitin through a highly solvated interface. Mutations that disrupt the interface inhibit MVB sorting, and the structure also explains how the TSG101 UEV can independently bind its ubiquitin and Pro-Thr/Ser-Ala-Pro peptide ligands. Remarkably, comparison with mapping data from other UEV and related E2 proteins indicates that although the different E2/UEV domains share the same structure and have conserved ubiquitin binding activity, they bind through very different interfaces.     

Up-regulation of tumor susceptibility gene 101 protein in ovarian carcinomas revealed by proteomics analyses

Small GTPase RAS plays a critical role in cellular signaling and oncogenic transformation. Proteomics analysis of genetically defined human ovarian cancer models identified the tumor susceptibility gene 101 (TSG101) as a downstream target of RAS oncogene. Mechanistic studies revealed a novel post-translational regulation of TSG101 through the RAS/RAF/MEK/MAPK signaling pathway and downstream molecules p14(ARF)/HDM2. Immunoanalysis using ovarian cancer samples and microtissue array revealed elevated TSG101 levels in human ovarian carcinomas. Silencing of TSG101 by short interfering RNA in ovarian cancer cells led to growth inhibition and cell death. Concurrent with the apparent growth-inhibitory effect, the levels of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1alpha (HIF-1alpha), as well as its cellular activity, were markedly reduced after TSG101 knockdown. These results demonstrate that TSG101 is important for CITED2- and HIF-1alpha-mediated cellular regulation in ovarian carcinomas.     

Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression

Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5') suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release. We observed that a mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release. We also determined the effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. We propose that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting.     

Abnormal regulation of TSG101 in mice with spongiform neurodegeneration

Spongiform neurodegeneration is characterized by the appearance of vacuoles throughout the central nervous system. It has many potential causes, but the underlying cellular mechanisms are not well understood. Mice lacking the E3 ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) develop age-dependent spongiform encephalopathy. We identified an interaction between a “PSAP” motif in MGRN1 and the ubiquitin E2 variant (UEV) domain of TSG101, a component of the endosomal sorting complex required for transport I (ESCRT-I), and demonstrate that MGRN1 multimonoubiquitinates TSG101. We examined the in vivo consequences of loss of MGRN1 on TSG101 expression and function in the mouse brain. The pattern of TSG101 ubiquitination differed in the brains of wild-type mice and Mgrn1 null mutant mice: at 1 month of age, null mutant mice had less ubiquitinated TSG101, while in adults, mutant mice had more ubiquitinated, insoluble TSG101 than wild-type mice. There was an associated increase in epidermal growth factor receptor (EGFR) levels in mutant brains. These results suggest that loss of MGRN1 promotes ubiquitination of TSG101 by other E3s and may prevent its disassociation from endosomal membranes or cause it to form insoluble aggregates. Our data implicate loss of normal TSG101 function in endo-lysosomal trafficking in the pathogenesis of spongiform neurodegeneration in Mgrn1 null mutant mice.     

GISP binding to TSG101 increases GABA receptor stability by down-regulating ESCRT-mediated lysosomal degradation

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABA(B) receptor subunit GABA(B1). Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA(B2) subunit, their co-expression in HEK293 cells increases levels of GABA(B2). GISP also inhibits TSG101-dependent GABA(B2) down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA(B2) degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.     

Consistent effects of TSG101 genetic variability on multiple outcomes of exposure to human immunodeficiency virus type 1

Tumor susceptibility gene 101 (TSG101) encodes a host cellular protein that is appropriated by human immunodeficiency virus type 1 (HIV-1) in the budding process of viral particles from infected cells. Variation in the coding or noncoding regions of the gene could potentially affect the degree of TSG101-mediated release of viral particles. While the coding regions of the gene were found to lack nonsynonymous variants, two polymorphic sites in the TSG101 5' area were identified that were associated with the rate of AIDS progression among Caucasians. These single-nucleotide polymorphisms (SNPs), located at positions -183 and +181 relative to the translation start, specify three haplotypes termed A, B, and C, which occur at frequencies of 67%, 21%, and 12%, respectively. Haplotype C is associated with relatively rapid AIDS progression, while haplotype B is associated with slower disease progression. Both effects were dominant over the intermediate haplotype A. The haplotypes also demonstrated parallel effects on the rate of CD4 T-cell depletion and viral load increase over time, as well as a possible influence on HIV-1 infection. The data raise the hypothesis that noncoding variation in TSG101 affects the efficiency of TSG101-mediated release of viral particles from infected cells, thereby altering levels of plasma viral load and subsequent disease progression.     

Hyaluronidase and collagenase inhibitory activities of the herbal formulation <Emphasis Type="Italic">Triphala guggulu</Emphasis>

Myrrh (guggulu) oleoresin from the Commiphora mukul tree is an important component of antiarthritic drugs in Ayurvedic medicine. Clinical data suggest that elevated levels of hyaluronidase and collagenase type 2 enzymes contribute significantly to cartilage degradation. Triphala guggulu (TG) is a guggulu-based formulation used for the treatment of arthritis. We assessed the chondroprotective potential of TG by examining its effects on the activities of pure hyaluronidase and collagenase type 2 enzymes. Triphala shodith guggulu (TSG), an intermediate in the production of TG, was also examined. A spectrophotometric method was used to assay Hyaluronidase activity, and to detect potential Hyaluronidase inhibitors. Aqueous and hydro-alcoholic extracts of TSG showed weak but dose-dependent inhibition of hyaluronidase activity. In contrast, the TG formulation was 50 times more potent than the TSG extract with respect to hyaluronidase inhibitory activity. A validated X-ray film-based assay was used to measure the gelatinase activity of pure collagenase type 2. Hydro-alcoholic extracts of the TG formulation were 4 times more potent than TSG with respect to collagenase inhibitory activity. Components of Triphala were also evaluated for their inhibitory activities on hyaluronidase and collagenase. This is the first report to show that the T2 component of Triphala (T. chebula) is a highly potent hyaluronidase and collagenase inhibitor. Thus, the TG formulation inhibits two major enzymes that can degrade cartilage matrix. Our study provides the first in vitro preclinical evidence of the chondroprotective properties of TG.     

Tillering and small grain 1 dominates the tryptophan aminotransferase family required for local auxin biosynthesis in rice

Auxin is a crucial phytohormone, controlling multiple aspects of plant growth and responses to the changing environment. However, the role of local auxin biosynthesis in specific developmental programs remains unknown in crops. This study characterized the rice tillering and small grain 1 (tsg1) mutant, which has more tillers but a smaller panicle and grain size resulting from a reduction in endogenous auxin. TSG1 encodes a tryptophan aminotransferase that is allelic to the FISH BONE (FIB) gene. The tsg1 mutant showed hypersensitivity to indole‐3‐acetic acid and the competitive inhibitor of aminotransferase, L‐kynurenine. TSG1 knockout resulted in an increased tiller number but reduction in grain number and size, and decrease in height. Meanwhile, deletion of the TSG1 homologs OsTAR1, OsTARL1, and OsTARL2 caused no obvious changes, although the phenotype of the TSG1/OsTAR1 double mutant was intensified and infertile, suggesting gene redundancy in the rice tryptophan aminotransferase family. Interestingly, TSG1 and OsTAR1, but not OsTARL1 and OsTARL2, displayed marked aminotransferase activity. Meanwhile, subcellular localization was identified as the endoplasmic reticulum, while phylogenetic analysis revealed functional divergence of TSG1 and OsTAR1 from OsTARL1 and OsTARL2. These findings suggest that TSG1 dominates the tryptophan aminotransferase family, playing a prominent role in local auxin biosynthesis in rice.     

TSG101 expression in gynecological tumors: relationship to cyclin D1, cyclin E, p53 and p16 proteins.

Recent studies have shown that in vitro steady-state expression of the tumor susceptibility gene TSG101 is important for maintenance of genomic stability and cell cycle regulation. To determine the contribution of TSG101 expression in neoplastic formation, expression of TSG101 protein levels were evaluated in primary ovarian and endometrial adenocarcinoma tumors. Expression of TSG101 was also examined in various tumor cell lines (PA-1, AN3CA, HeLa, HS578T, HCT116). Full-length TSG101 protein was detected in these tumors and cell lines indicating that intragenic deletions were not characteristic of TSG101. In addition, TSG101 protein levels were compared with aberrations of prominent cell cycle regulatory molecules such as cyclin D1, cyclin E, p16 and p53. Reduced TSG101 protein was observed in 36% (8/22) of ovarian and 17% (1/6) of endometrial adenocarcinoma. Aberrant levels of p53, p16, cyclin D or E were comparable to published studies indicating that the clinicopathological distribution of these cases did not favor advanced stage tumors. Altogether, these findings suggest that a down-regulation of TSG101 is associated with tumorigenesis in a subgroup of gynecological tumors.