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

The 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.

Sp100A promotes chromatin decondensation at a cytomegalovirus-promoter–regulated transcription site

Promyelocytic leukemia nuclear bodies (PML-NBs)/nuclear domain 10s (ND10s) are nuclear structures that contain many transcriptional and chromatin regulatory factors. One of these, Sp100, is expressed from a single-copy gene and spliced into four isoforms (A, B, C, and HMG), which differentially regulate transcription. Here we evaluate Sp100 function in single cells using an inducible cytomegalovirus-promoter–regulated transgene, visualized as a chromatinized transcription site. Sp100A is the isoform most strongly recruited to the transgene array, and it significantly increases chromatin decondensation. However, Sp100A cannot overcome Daxx- and α-thalassemia mental retardation, X-linked (ATRX)–mediated transcriptional repression, which indicates that PML-NB/ND10 factors function within a regulatory hierarchy. Sp100A increases and Sp100B, which contains a SAND domain, decreases acetyl-lysine regulatory factor levels at activated sites, suggesting that Sp100 isoforms differentially regulate transcription by modulating lysine acetylation. In contrast to Daxx, ATRX, and PML, Sp100 is recruited to activated arrays in cells expressing the herpes simplex virus type 1 E3 ubiquitin ligase, ICP0, which degrades all Sp100 isoforms except unsumoylated Sp100A. The recruitment Sp100A(K297R), which cannot be sumoylated, further suggests that sumoylation plays an important role in regulating Sp100 isoform levels at transcription sites. This study provides insight into the ways in which viruses may modulate Sp100 to promote their replication cycles.