scholarly journals The AIRE G228W mutation results in a longer-lasting AIRE-SIRT1 interaction

2021 ◽  
Author(s):  
Jadson C. Santos ◽  
Mariangela Dametto ◽  
Ana Paula Masson ◽  
Vitor M. Faça ◽  
Rodrigo Bonacin ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Thymic Epithelial Cells ◽  
Physical Interaction ◽  
Wild Type ◽  
Loss Of Self ◽  
Wild Type Counterpart ◽  
Sand Domain ◽  
Medullary Thymic Epithelial Cells ◽  
Protein Binding Kinetics

AbstractThe in silico and in vitro binding of a peptide covering a part of the autoimmune regulator (AIRE) SAND domain with the SIRT1 protein provides a powerful model system for studying the mechanism of the dominant SAND G228W mutation, which is the causative of APS-1 autoimmune syndrome. It is known that the mutant G228W AIRE protein accumulates more within the nucleus of cells than its wild-type counterpart does. This accumulation is not physiological and is associated with loss of AIRE function. However, the precise molecular mechanism that leads to AIRE accumulation is not yet known. AIRE works as a tetramer and interacts with partner proteins to form the “AIRE complex” that pushes RNA Pol II stalling in the chromatin of medullary thymic epithelial cells. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates Lys residues of the SAND domain. Once AIRE is deacetylated, the binding with SIRT1 is undone, allowing the complex to proceed downstream. Here, we integrate molecular modeling, docking, dynamics, and surface plasmon resonance approaches to compare the structure and energetics of binding/release between AIRE G228 (wild-type) or W228 (mutant) peptides to SIRT1. We find that the proximity of G228W mutation to a K aminoacid residue in the SAND domain promotes a longer-lasting AIRE-SIRT1 interaction. The lasting interaction might cause a delay in the AIRE SAND domain to be released from the SIRT1 catalytic site, which might cause accumulation of the defective AIRE mutant protein in the nuclei of cells.SignificanceThis report reveals the mechanism of the pathogenic and dominant G228W mutation in the AIRE SAND domain. The G228W mutation is found in APS-1 syndrome patients, and it is critical to understand the molecular basis of loss of self-representation, a challenging aspect for immunology. Through modeling, molecular dynamics, and protein binding kinetics, we found that the G228W mutation leads to a stronger physical interaction between the AIRE SAND domain and the SIRT1 protein when compared to the equivalent wild-type segment. The short-term consequence of this stronger interaction is that the release of the AIRE-SIRT1 binding is slower. This might explain the reason that cells carrying the G228W mutation accumulate AIRE protein in their nuclei. This finding reveals with precision the AIRE-SIRT1 binding and the molecular mechanism of the human AIRE G228W mutation.

scholarly journals Unstable Spinocerebellar Ataxia Type 10 (ATTCT)·(AGAAT) Repeats Are Associated with Aberrant Replication at the ATX10 Locus and Replication Origin-Dependent Expansion at an Ectopic Site in Human Cells

2007 ◽  
Vol 27 (22) ◽  
pp. 7828-7838 ◽  
Author(s):  
Guoqi Liu ◽  
John J. Bissler ◽  
Richard R. Sinden ◽  
Michael Leffak
Keyword(s):  
Molecular Mechanism ◽  
Cell Lines ◽  
Spinocerebellar Ataxia ◽  
Replication Origin ◽  
Spinocerebellar Ataxia Type ◽  
Dna Unwinding ◽  
Wild Type ◽  
Normal Length ◽  
Population Doublings

ABSTRACT Spinocerebellar ataxia type 10 (SCA10) is associated with expansion of (ATTCT) n repeats (where n is the number of repeats) within the ataxin 10 (ATX10/E46L) gene. The demonstration that (ATTCT) n tracts can act as DNA unwinding elements (DUEs) in vitro has suggested that aberrant replication origin activity occurs at expanded (ATTCT) n tracts and may lead to their instability. Here, we confirm these predictions. The wild-type ATX10 locus displays inefficient origin activity, but origin activity is elevated at the expanded ATX10 loci in patient-derived cells. To test whether (ATTCT) n tracts can potentiate origin activity, cell lines were constructed that contain ectopic copies of the c-myc replicator in which the essential DUE was replaced by ATX10 DUEs with (ATTCT) n . ATX10 DUEs containing (ATTCT)27 or (ATTCT)48, but not (ATTCT)8 or (ATTCT)13, could substitute functionally for the c-myc DUE, but (ATTCT)48 could not act as an autonomous replicator. Significantly, chimeric c-myc replicators containing ATX10 DUEs displayed length-dependent (ATTCT) n instability. By 250 population doublings, dramatic two- and fourfold length expansions were observed for (ATTCT)27 and (ATTCT)48 but not for (ATTCT)8 or (ATTCT)13. These results implicate replication origin activity as one molecular mechanism associated with the instability of (ATTCT) n tracts that are longer than normal length.

HOXA11 Mutation in Amegakaryocytic Thrombocytopenia with Radio-Ulnar Synostosis Syndrome Inhibits Megakaryocytic Differentiation In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1751-1751 ◽  
Author(s):  
Regina D. Horvat-Switzer ◽  
Alexis A. Thompson
Keyword(s):  
Dna Binding ◽  
Point Mutation ◽  
Fold Increase ◽  
Physical Interaction ◽  
Wild Type ◽  
Mobility Shift ◽  
Transfected Cells ◽  
Megakaryocytic Differentiation ◽  
Dnase Treatment

Abstract Homeobox genes encode for regulatory proteins central to bone morphogenesis as well as hematopoietic differentiation and proliferation. Previously we have identified an inherited syndrome of congenital amegakaryocytic thrombocytopenia and radio-ulnar synostosis that is associated with a point mutation in the third helix of HOXA11 homeodomain (HOXA11-ΔH3). We postulated that this mutation results in a truncated protein with impaired DNA binding efficiency. Compared to wild type (HOXA11-WT) in vitro transcription and translation of HOXA11-ΔH3 produced a truncated form of the protein. Electropheretic mobility shift assays (EMSA) confirmed that product from a HoxA11-WT cDNA expression plasmid interacts with the DNA binding consensus sequence for HoxA11 and this interaction is most efficient when its TALE transcription co-factor, Meis1b, is also present. Using the truncated HOXA11-ΔH3 product, the mobility shift was abrogated even in the presence of Meis1b, suggesting the point mutant causes a disruption in the DNA binding capacity of HoxA11. We investigated whether the point mutation affected the physical protein-protein interaction that occurs between HoxA11 and Meis1b. Using GST-pulldown assays we examined these interactions. Meis1b showed a 11.8±4.3 fold increase in interaction with HOXA11-WT and a 13.7±3.6 fold increase with HOXA11-ΔH3 in the presence of DNA. DNAse treatment decreased the interactions to 6±0.24 and 7±1.1 for HOXA11-WT and -ΔH3 respectively. These data suggest the physical interaction between HoxA11 and Meis1b is a protein-protein association and that the presence of DNA may serve to stabilize this interaction. We next investigated the effects of HOXA11 on megakaryocytic differentiation. The human erythroleukemia cell line, K562, was stably transfected with expression vectors for either HOXA11-WT or -ΔH3. The transfected cell lines were then treated with the pharmacological agents, phorbol-12 myristate-13 acetate (PMA) or staurosporine (STSP), to induce megakaryocytic differentiation. Both agents induce surface expression of the megakaryocytic/platelet-specific antigen, CD61 and cause morphological changes. CD61 expression in untransfected cells increased from 1.6±0.4% at baseline to 91±0.5% with PMA and to 81±2.4% when treated with STSP. Marked differences in CD61 expression and viability were seen in transfected cells. HOXA11-WT and -ΔH3 lead to a significantly less CD61 expression, 53±10% and 69.8±5.8% respectively following PMA induction, compared to untransfected cells. PMA treatment of cells that constitutively express HOXA11-WT or -ΔH3 resulted in a marked reduction in cell viability and suggests that the normal silencing of HOXA11 in early hematopoiesis may be an essential aspect of its function. Neither HOXA11-WT nor -ΔH3 cells showed an increase in expression of CD61 expression following STSP treatment, 10.1±5.9% and 3.7±2.4% respectively. Interestingly, nearly all transfected cells treated with STSP remained viable, suggesting divergent signaling pathways are utilized by this differentiating agent. There were no substantial differences between wild type and mutant HOXA11 with either inducing agent, suggesting that inhibition of differentiation by HOXA11 involves other non-homeodomain regulatory sequences of this gene. Taken together these data begin to give us insight into the molecular mechanisms by which HoxA11 regulates megakaryocytic differentiation.

Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells

2011 ◽  
Vol 106 (10) ◽  
pp. 693-704 ◽  
Author(s):  
Valeria Bozzi ◽  
Emanuele Panza ◽  
Serena Barozzi ◽  
Cristian Gruppi ◽  
Marco Seri ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Cell Types ◽  
Type I ◽  
Wild Type ◽  
Mutant Proteins ◽  
Wild Type Counterpart ◽  
Platelet Release ◽  
Myosin Iia ◽  
Different Cell Types

SummaryMYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for the heavy chain of nonmuscle myosin-IIA (NMMHC-IIA). Recent in vitro studies led to the hypothesis that thrombocytopenia of MYH9-RD derives from an ectopic platelet release by megakaryocytes in the osteoblastic areas of bone marrow (BM), which are enriched in type I collagen, rather than in vascular spaces. SDF-1-driven migration of megakaryocytes within BM to reach the vascular spaces is a key mechanism for platelet biogenesis. Since myosin-IIA is implicated in polarised migration of different cell types, we hypothesised that MYH9 mutations could interfere with this mechanism. We therefore investigated the SDF-1-driven migration of a megakaryoblastic cell line, Dami cells, on type I collagen or fibrinogen by a modified transwell assay. Inhibition of myosin-IIA ATPase activity suppressed the SDF-1-driven migration of Dami cells, while over-expression of NMMHC-IIA increased the efficiency of chemotaxis, indicat- ing a role for NMMHC-IIA in this mechanism. Transfection of cells with three MYH9 mutations frequently responsible for MYH9-RD (p.R702C, p.D1424H, or p.R1933X) resulted in a defective SDF-1-driven migration with respect to the wild-type counterpart and in increased cell spreading onto collagen. Analysis of differential localisation of wild-type and mutant proteins suggested that mutant NMMHC-IIAs had an impaired cytoplasmic re-organisation in functional cytoskeletal structures after cell adhesion to collagen. These findings support the hypothesis that a defect of SDF-1-driven migration of megakaryocytes induced by MYH9 mutations contributes to ectopic platelet release in the BM osteoblastic areas, resulting in ineffective platelet production.

scholarly journals HIV-1 Vpr Enhances Viral Burden by Facilitating Infection of Tissue Macrophages but Not Nondividing CD4+ T Cells

2001 ◽  
Vol 194 (10) ◽  
pp. 1407-1419 ◽  
Author(s):  
Daniel A. Eckstein ◽  
Michael P. Sherman ◽  
Michael L. Penn ◽  
Peggy S. Chin ◽  
Carlos M.C. De Noronha ◽  
...  
Keyword(s):  
T Cells ◽  
Lymphoid Tissue ◽  
Wild Type ◽  
Cellular Targets ◽  
Viral Burden ◽  
Immunodeficiency Virus ◽  
Wild Type Counterpart ◽  
Cc Chemokine Receptor 5 ◽  
Hiv 1

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.

scholarly journals A Germline Mutation in the POT1 Gene Is a Candidate for Familial Non-Medullary Thyroid Cancer

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1441 ◽  
Author(s):  
Aayushi Srivastava ◽  
Beiping Miao ◽  
Diamanto Skopelitou ◽  
Varun Kumar ◽  
Abhishek Kumar ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Germline Mutation ◽  
Medullary Thyroid Cancer ◽  
Current Knowledge ◽  
Wild Type ◽  
Medullary Thyroid ◽  
Wild Type Counterpart ◽  
In Silico Analyses ◽  
Protection Of Telomeres 1

Non-medullary thyroid cancer (NMTC) is a common endocrine malignancy with a genetic basis that has yet to be unequivocally established. In a recent whole-genome sequencing study of five families with occurrence of NMTCs, we shortlisted promising variants with the help of bioinformatics tools. Here, we report in silico analyses and in vitro experiments on a novel germline variant (p.V29L) in the highly conserved oligonucleotide/oligosaccharide binding domain of the Protection of Telomeres 1 (POT1) gene in one of the families. The results showed a reduction in telomere-bound POT1 levels in the mutant protein as compared to its wild-type counterpart. HEK293T cells carrying POT1 p.V29L showed increased telomere length in comparison to wild-type cells, suggesting that the mutation causes telomere dysfunction and may play a role in predisposition to NMTC in this family. While one germline mutation in POT1 has already been reported in a melanoma-prone family with prevalence of thyroid cancers, we report the first of such mutations in a family affected solely by NMTCs, thus expanding current knowledge on shelterin complex-associated cancers.

scholarly journals Autoimmune regulator act in synergism with thymocyte adhesion in the control of lncRNAs in medullary thymic epithelial cells

2021 ◽  
Author(s):  
Max J Duarte ◽  
Romario S Mascarenhas ◽  
Amanda F Assis ◽  
Pedro P Tanaka ◽  
Cesar A Speck-Hernandez ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Model Systems ◽  
Thymic Epithelial Cells ◽  
Large Set ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Autoimmune Regulator ◽  
Medullary Thymic Epithelial Cells

The autoimmune regulator (Aire) gene in medullary thymic epithelial cells (mTECs) encodes the AIRE protein, which interacts with its partners within the nucleus. This Aire complex induces stalled RNA Pol II on chromatin to proceed with transcription elongation of a large set of messenger RNAs and microRNAs. Considering that RNA Pol II also transcribes long noncoding RNAs (lncRNAs), we hypothesized that Aire might be implicated in the upstream control of this RNA species. To test this, we employed a loss-of-function approach in which Aire knockout mTECs were compared to Aire wild-type mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. RNA sequencing enables the differential expression profiling of lncRNAs when these cells adhere in vitro to thymocytes or do not adhere to them as a way to test the effect of cell adhesion. Sets of lncRNAs that are unique and that are shared in vitro and in vivo were identified. Among these, we found the Aire-dependent lncRNAs as for example, Platr28, Ifi30, Morrbid, Malat1, and Xist. This finding represents the first evidence that Aire mediates the transcription of lncRNAs in mTECs. Microarray hybridizations enabled us to observe that temporal thymocyte adhesion modulates the expression levels of such lncRNAs as Morrbid, Xist, and Fbxl12o after 36h of adhesion. This finding shows the existence of a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs that might be important for immune self-representation.

scholarly journals Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

2021 ◽  
Author(s):  
Catherine A. A. Beauchemin ◽  
Eric G. Paradis ◽  
Lady Tatiana Pinilla ◽  
Benjamin P. Holder ◽  
Yacine Abed ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mathematical Modelling ◽  
Wild Type ◽  
Oseltamivir Resistance ◽  
Wild Type Counterpart ◽  
Experimental Variability ◽  
Infected Cells ◽  
Eclipse Phase ◽  
H275y Mutation

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.

Arsenic Binding to RBCC Domain Modulates Oncogenic PML-RARa and Wild-Type PML as a Key Molecular Mechanism of Arsenic Trioxide Treatment for APL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 592-592
Author(s):  
Xiaowei Zhang ◽  
Xiaojing Yan ◽  
Feifei Yang ◽  
Ziren Zhou ◽  
Ziyu Wu ◽  
...  
Keyword(s):  
Conformational Change ◽  
Molecular Mechanism ◽  
Metal Binding ◽  
Conflicts Of Interest ◽  
Periodic Fever ◽  
Coiled Coil ◽  
Wild Type ◽  
Selective Degradation ◽  
Enzymatic Machinery

Abstract Abstract 592 Arsenicals represent one group of the oldest drugs used in both traditional Chinese medicine (TCM) and Western medicine since 2,000 years ago to treat a variety of ailments from periodic fever to cancer. Recently, this ancient remedy has been revived due to its remarkable therapeutic efficacy for acute promyelocytic leukemia (APL) through selective degradation of the leukemogenic PML-RARaƒn as well as the wild-type PML protein. However, the precise molecular mechanism leading to arsenic-initiated modulationƒn of the target proteins remained unclear. Here we show that arsenic directly binds to PML and PML-RARaƒn through their RBCC (RING-B box-coiled coil) domain which contains conserved cysteine/histidine residues with metal-binding ability. Among RBCC domain, the RING and B2 motif are responsible for arsenic binding in cells, with recombinant RING motif showing the highest affinity to arsenic binding in vitro. We also observed that arsenic tends to coordinate with three sulfur atoms from the three conserved cysteines in the RING zinc finger. Arsenic binding alters the native structure of RING coordinated with zinc and induces its oligomerization through arsenic-mediated conformational change by intramolecular coordination as well as cross-linking between two RING motifs. Following conformational change and oligomerization of PML RBCC with arsenic binding, PML and PML-RARa undergoes SUMOylation through enhanced interaction with Ubc9, the E2 ligase for SUMOylation. Our findings provide the evidence that the PML RBCC domain is the direct target of arsenic and that the structural change of PML RBCC induced by arsenic binding facilitates the enhanced interaction with the cellular enzymatic machinery for protein SUMOylation/ubiquitination, which ultimately leads to the degradation of PML-RARa and cell differentiation and/or apoptosis. This mechanism sheds new insights into the mechanism of action of As2O3 for APL treatment, a model of targeted cancer therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells

2021 ◽  
Author(s):  
Pedro P Tanaka ◽  
Ernna H Oliveira ◽  
Mayara C Machado ◽  
Max J Duarte ◽  
Amanda F Assis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Large Scale ◽  
Interaction Analysis ◽  
Luciferase Reporter ◽  
Thymic Epithelial Cells ◽  
Posttranscriptional Control ◽  
Autoimmune Regulator ◽  
Aire Gene ◽  
Medullary Thymic Epithelial Cells

Background: The autoimmune regulator (Aire) gene is critical for the appropriate establishment of central immune tolerance. As one of the main controllers of promiscuous gene expression in the thymus, Aire promotes the expression of thousands of downstream tissue-restricted antigen (TRA) genes, cell adhesion genes and transcription factor genes in medullary thymic epithelial cells (mTECs). Despite the increasing knowledge about the role of Aire as an upstream transcriptional controller, little is known about the mechanisms by which this gene could be regulated. Results: Here, we assessed the posttranscriptional control of Aire by miRNAs. The in silico miRNA-mRNA interaction analysis predicted thermodynamically stable hybridization between the 3UTR of Aire mRNA and miR-155, which was confirmed to occur within the cellular milieu through a luciferase reporter assay. This finding enabled us to hypothesize that miR-155 might play a role as an intracellular posttranscriptional regulator of Aire mRNA. To test this hypothesis, we transfected a murine mTEC cell line with a miR-155 mimic in vitro, which reduced the mRNA and protein levels of Aire. Moreover, large-scale transcriptome analysis showed the modulation of 311 downstream mRNAs, which included 58 TRA mRNAs. Moreover, miR-155 mimic-transfected cells exhibited a decrease in their chemotaxis property compared with control thymocytes. Conclusion: Overall, the results indicate that miR-155 may posttranscriptionally control Aire mRNA as well as a crucial process by which mTECs allow migration of thymocytes through chemotaxis.

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