Trypanosoma Brucei Telomere Functions in Antigenic Variation

Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and Nagana in cattle. They evade the host\u27s immune defense by periodically switching their major surface antigen, variant surface glycoprotein (VSG), a phenomenon termed antigenic variation. Inside its mammalian host, bloodstream form (BF) T. brucei monoallelically expresses its major surface molecule VSG from the VSG Expression Sites (ESs) located at subtelomeric loci. Monoallelic VSG expression ensures effective antigenic variation and maximizes the efficiency of T. brucei pathogenesis. In the mid-gut of its insect host (tsetse), procyclic form (PF) T. brucei expresses procyclins as the major surface molecules and all VSGs are silent. After the migration to the salivary glands of the tsetse fly, T. brucei cells differentiate into metacyclic forms and express metacyclic VSGs (mVSGs). Therefore, VSG silencing is important for the normal development of T. brucei. Telomeres are important for the regulation of antigenic variation. TbRAP1 was previously identified as an intrinsic component of the T. brucei telomere complex and was shown to be important for ES-linked VSG silencing in BF cells. Our studies further established that TbRAP1 is essential for cell proliferation and required for VSG silencing in PF cells. Apart form ES-linked VSGs, TbRAP1 also regulates the silencing of mVSGs in both BF and PF cells. The strength of TbRAP1 mediated VSG silencing is stronger in PF cells compared to that in the BF cells. In addition, the TbRAP1-mediated VSG silencing in PF cells involves chromatin remodeling. TbTRF, a duplex telomere DNA binding protein that interacts with TbRAP1, does not affect VSG silencing but regulates telomere structure. TbTRFH (TRF homology domain) is required for the homodimerization of TbTRF as well as for interaction with TbRAP1. We established several TbTRFH mutants and determined the critical regions required for homodimerization of TbTRF by performing yeast two-hybrid analysis. TERRA (telomeric repeat- c

Trypanosoma Brucei Telomere Functions in Antigenic Variation

Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and Nagana in cattle. They evade the host\u27s immune defense by periodically switching their major surface antigen, variant surface glycoprotein (VSG), a phenomenon termed antigenic variation. Inside its mammalian host, bloodstream form (BF) T. brucei monoallelically expresses its major surface molecule VSG from the VSG Expression Sites (ESs) located at subtelomeric loci. Monoallelic VSG expression ensures effective antigenic variation and maximizes the efficiency of T. brucei pathogenesis. In the mid-gut of its insect host (tsetse), procyclic form (PF) T. brucei expresses procyclins as the major surface molecules and all VSGs are silent. After the migration to the salivary glands of the tsetse fly, T. brucei cells differentiate into metacyclic forms and express metacyclic VSGs (mVSGs). Therefore, VSG silencing is important for the normal development of T. brucei. Telomeres are important for the regulation of antigenic variation. TbRAP1 was previously identified as an intrinsic component of the T. brucei telomere complex and was shown to be important for ES-linked VSG silencing in BF cells. Our studies further established that TbRAP1 is essential for cell proliferation and required for VSG silencing in PF cells. Apart form ES-linked VSGs, TbRAP1 also regulates the silencing of mVSGs in both BF and PF cells. The strength of TbRAP1 mediated VSG silencing is stronger in PF cells compared to that in the BF cells. In addition, the TbRAP1-mediated VSG silencing in PF cells involves chromatin remodeling. TbTRF, a duplex telomere DNA binding protein that interacts with TbRAP1, does not affect VSG silencing but regulates telomere structure. TbTRFH (TRF homology domain) is required for the homodimerization of TbTRF as well as for interaction with TbRAP1. We established several TbTRFH mutants and determined the critical regions required for homodimerization of TbTRF by performing yeast two-hybrid analysis. TERRA (telomeric repeat- c

Trypanosoma brucei RAP1 Maintains Telomere and Subtelomere Integrity by Suppressing TERRA and Telomeric RNA: DNA Hybrids

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host\u27s immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversionmediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription

Trypanosoma Brucei Telomere Functions in Antigenic Variation

Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and Nagana in cattle. They evade the host\u27s immune defense by periodically switching their major surface antigen, variant surface glycoprotein (VSG), a phenomenon termed antigenic variation. Inside its mammalian host, bloodstream form (BF) T. brucei monoallelically expresses its major surface molecule VSG from the VSG Expression Sites (ESs) located at subtelomeric loci. Monoallelic VSG expression ensures effective antigenic variation and maximizes the efficiency of T. brucei pathogenesis. In the mid-gut of its insect host (tsetse), procyclic form (PF) T. brucei expresses procyclins as the major surface molecules and all VSGs are silent. After the migration to the salivary glands of the tsetse fly, T. brucei cells differentiate into metacyclic forms and express metacyclic VSGs (mVSGs). Therefore, VSG silencing is important for the normal development of T. brucei. Telomeres are important for the regulation of antigenic variation. TbRAP1 was previously identified as an intrinsic component of the T. brucei telomere complex and was shown to be important for ES-linked VSG silencing in BF cells. Our studies further established that TbRAP1 is essential for cell proliferation and required for VSG silencing in PF cells. Apart form ES-linked VSGs, TbRAP1 also regulates the silencing of mVSGs in both BF and PF cells. The strength of TbRAP1 mediated VSG silencing is stronger in PF cells compared to that in the BF cells. In addition, the TbRAP1-mediated VSG silencing in PF cells involves chromatin remodeling. TbTRF, a duplex telomere DNA binding protein that interacts with TbRAP1, does not affect VSG silencing but regulates telomere structure. TbTRFH (TRF homology domain) is required for the homodimerization of TbTRF as well as for interaction with TbRAP1. We established several TbTRFH mutants and determined the critical regions required for homodimerization of TbTRF by performing yeast two-hybrid analysis. TERRA (telomeric repeat- c

Silencing Subtelomeric VSGs by Trypanosoma brucei RAP1 at the Insect Stage Involves Chromatin Structure Changes

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen variant surface glycoprotein (VSG) to evade mammalian host immune responses at the bloodstream form (BF) stage. Monoallelic expression of BF Expression Site (BES)-linked VSGs and silencing of metacyclic VSGs (mVSGs) in BF cells are essential for antigenic variation, whereas silencing of both BES-linked and mVSGs in the procyclic form (PF) cells is important for cell survival in the midgut of its insect vector. We have previously shown that silencing BES-linked VSGs in BF cells depends on TbRAP1. We now show that TbRAP1 silences both BES-linked and mVSGs at both BF and PF stages. The strength of TbRAP1-mediated BES-linked VSG silencing is stronger in the PF cells than that in BF cells. In addition, Formaldehyde-Assisted Isolation of Regulatory Elements analysis and MNase digestion demonstrated that depletion of TbRAP1 in PF cells led to a chromatin structure change, which is significantly stronger at the subtelomeric VSG loci than at chromosome internal loci. On the contrary, no significant chromatin structure changes were detected on depletion of TbRAP1 in BF cells. Our observations indicate that TbRAP1 helps to determine the chromatin structure at the insect stage, which likely contributes to its strong silencing effect on VSGs. © 2013 The Author(s)

The Biophysical Interaction of the Danger-Associated Molecular Pattern (DAMP) Calreticulin with the Pattern-Associated Molecular Pattern (PAMP) Lipopolysaccharide

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity

TGF-β activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth

<p>We previously reported that aberrant TGF-β/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27^kip1(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-β signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-β increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-β-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-β signaling.</p

Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen variant surface glycoprotein (VSG) to evade mammalian host immune responses at the bloodstream form (BF) stage. Monoallelic expression of BF Expression Site (BES)-linked VSGs and silencing of metacyclic VSGs (mVSGs) in BF cells are essential for antigenic variation, whereas silencing of both BES-linked and mVSGs in the procyclic form (PF) cells is important for cell survival in the midgut of its insect vector. We have previously shown that silencing BES-linked VSGs in BF cells depends on TbRAP1. We now show that TbRAP1 silences both BES-linked and mVSGs at both BF and PF stages. The strength of TbRAP1-mediated BES-linked VSG silencing is stronger in the PF cells than that in BF cells. In addition, Formaldehyde-Assisted Isolation of Regulatory Elements analysis and MNase digestion demonstrated that depletion of TbRAP1 in PF cells led to a chromatin structure change, which is significantly stronger at the subtelomeric VSG loci than at chromosome internal loci. On the contrary, no significant chromatin structure changes were detected on depletion of TbRAP1 in BF cells. Our observations indicate that TbRAP1 helps to determine the chromatin structure at the insect stage, which likely contributes to its strong silencing effect on VSGs