Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription

Abstract

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.

LPS induces GFAT2 expression to promote O-GlcNAcylation and attenuate inflammation in macrophages

“…Moreover, using a luminescent assay [33,42], we found that LPS treatment had no detectable effect on OGT activity in cell extracts from RAW264.7 cells (Fig. 2D).…”

“…After 3 washes, the precipitated proteins were submitted to an additional wash in OGT assay buffer containing 50 mM Tris-HCl and 12.5 mM MgCl2, pH7.5. OGT assay was then performed on protein-G sepharose bound OGT [33] using the bioluminescent UDP-Glo TM glycosyltransferase assay (Promega) exactly as described in the manufacturer instructions [42].…”

“…Sub-cellular relocalisation of OGT in different cell compartments has been observed upon stimulation of membrane receptors [47,48], resulting in compartment specific changes in O-GlcNAcylation activity. In order to evaluate whether TLR4 stimulation affects protein O-GlcNAcylation in different cellular compartments in macrophages, we used BRET-based O-GlcNAc biosensors [33]. These biosensors are composed of Rluc8 luciferase fused to a lectin domain (GafD), a known OGT substrate peptide derived from casein kinase II, followed by the Venus variant of the yellow fluorescent protein (Fig.…”

Iron down-regulates leptin by suppressing protein O-GlcNAc modification in adipocytes, resulting in decreased levels of O-glycosylated CREB

“…O-GlcNAcylation of CREB was originally identified in the brain (31), where its glycosylation is involved in memory formation (32). The functional consequences of CREB glycosylation have since been demonstrated in nonneuronal tissues as well (33), although it remains little studied. Crosstalk between phosphorylation and O-GlcNAcylation of CREB, however, has been shown to modulate its activity (34).…”

Critical observations that shaped our understanding of the function(s) of intracellular glycosylation (O‐GlcNAc)

“…Numerous interactors have been identified including fatty acid synthase, whose association inhibits OGA activity . OGA is often found in a complex with OGT ; the association of these two proteins with proteins such as the GATA‐1·FOG‐1·Mi2β repressor complex, targets O‐GlcNAc cycling to promoters . OGA and OGT are also targeted to midbodies in complex with Aurora B Kinase and Protein Phosphatase 1.…”