Dynamic glycosylation of the transcription factor CREB: A potential role in gene regulation

We report that CREB (cyclic AMP-responsive element-binding protein), a transcription factor essential for long-term memory, is O-GlcNAc glycosylated in the mammalian brain. Glycosylation occurs at two sites within the Q2 domain and disrupts the interaction between CREB and TAF_(II)130, thereby repressing the transcriptional activity of CREB in vitro. These findings have important implications for the role of O-GlcNAc glycosylation in gene regulation, and they provide a link between O-GlcNAc and information storage processes in the brain

A Chemoenzymatic Approach toward the Rapid and Sensitive Detection of O-GlcNAc Posttranslational Modifications

We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of β-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications

Identification and Functional Analysis of O-G1cNAc Glycosylation on the Transcription Factor cAMP-Response Element Binding Protein

The survival and development of organisms requires the ability of cells to communicate with the environment and with surrounding cells. This demand has led to the evolution of a number of methods used for communication. Chief among these is the ability to modify protein function with post-translational modifications (PTMs). PTMs allow cells to use a single protein for a variety of tasks and link protein activity with a specific environmental or cellular cue. Modification of transcription factors has arisen as a key model for the study of PTMs and their effects on cell processes. PTMs modulate transcriptional activity required for key processes such as development, differentiation and cell survival. The eukaryotic transcription factor cAMP-response element binding protein (CREB) is a transcription factor that confers dynamic control of a number of cellular processes including neuronal and pancreatic cell survival, gluconeogenesis and neuronal long-term potentiation. CREB is activated by phosphorylation of single serine residue. The observation that a number of kinase signaling cascades converge on CREB has led to the question of how cells deal with the apparent loss of signal identity that occurs as a result of this convergence. In this thesis I describe the identification, characterization and functional analysis of a novel PTM of CREB, O-GlcNAc glycosylation, that provides an additional level of control of CREB activity. CREB glycosylation moderates phosphorylation-dependent CREB activity and reduces CREB-dependent gene expression in pancreatic [beta]-cells, and as a result promotes [beta]-cell death, as observed in type II diabetes. CREB glycosylation offers us an example of how cells use multiple PTMs to control protein function and how dysfunction in the regulation of these modifications may contribute to disease states.</p

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old