HDAC11 regulates type I interferon signaling through defatty-acylation of SHMT2

The smallest histone deacetylase (HDAC) and the only class IV HDAC member, HDAC11, is reported to regulate immune activation and tumorigenesis, yet its biochemical function is largely unknown. Here we identify HDAC11 as an efficient lysine defatty-acylase that is >10,000-fold more efficient than its deacetylase activity. Through proteomics studies, we hypothesized and later biochemically validated SHMT2 as a defatty-acylation substrate of HDAC11. HDAC11-catalyzed defatty-acylation did not affect the enzymatic activity of SHMT2. Instead, it affects the ability of SHMT2 to regulate type I IFN receptor ubiquitination and cell surface level. Correspondingly, HDAC11 depletion increased type I IFN signaling in both cell culture and mice. This study not only demonstrates that HDAC11 has an activity that is much more efficient than the corresponding deacetylase activity, but also expands the physiological functions of HDAC11 and protein lysine fatty acylation, which opens up opportunities to develop HDAC11-specific inhibitors as therapeutics to modulate immune responses.

Abstract 130: Arginine Methylation of Smad6 Defines Bone-morphogenetic-protein (BMP) Signaling Duration and Intensity

Bone-morphogenetic-protein (BMP)/Smads signaling pathway plays crucial role during heart development and vessel angiogenesis. BMP signaling is induced by the binding of BMP ligands (eg. BMP4) to their receptors, which recruit and phosphorylate receptor-Smads (R-Smads, eg. Smad1, Smad5) that form nuclear-transporting complexes with Smad4 for transcriptional regulation. Smad6 is an inhibitory Smad expresses predominantly in atria-ventricular cushion and outflow tract of the developing mouse heart, and expands to valves and great vessels. At the cell surface level, Smad6 binds to BMP type I receptor to block R-Smads recruitment to the receptor. At cytosolic level, Smad6 block Smad1/Smad4 complex formation. In the nucleus, Smad6 represses transcription. How these three levels of regulation are coordinated to inhibit BMP signaling is not known. We previously showed that BMP ligand induces an acute Smad6 methylation at arginine 74 (R74) at the cell surface level by a methyltransferase PRMT1, and methyl-Smad6 dissociates from receptor to allow receptor-induced Smad1/5 phosphorylation and activation. We further identified a delayed methylation on arginine 81 (R81) of Smad6 in the cytosol by PRMT1. We found that R81 methylation is required for BMP signaling-induced recruitment of Smad6 to phosphor-Smad1; it is also required for Smad6 to disrupt phosphor-Smad1/Smad4 complex formation and the following nuclear transportation, as well as for Smad6 to suppress Smad1 targeting gene transactivation. Previous findings indicate that Smad6 binds to type I receptor and Smad1 through its C-terminal region. We examined how arginine methylation in the N-terminal region, regulates the binding properties of C-terminal Smad6. We found that N-terminal Smad6 stabilizes the interaction between C-terminal Smad6 and Smad1 and enhances Smad6 inhibitory function. Disruption of R81 methylation results in loss of inhibitory function because of an increase in binding between N-term and C-term Smad6 that results in a "closed" conformation. In summary, R81 methylation controls Smad6 activity and R81 methylation of Smad6 defines the duration and intensity of BMP-induced Smad1/5 signaling.

Abstract 323: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase

Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that apoAI can solubilize liposomes made with egg PC:PS (8:2) only at acidic pH, where apoAI has unfolded to expose more hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation. Methods and Results: We found that apoAI unfolding in guanidine was facilitated at pH 5.0 vs. pH 7.5 (EC50 = 0.75 M and 0.94 M guanidine, respectively). We confirmed that apoAI solubilization of and binding to POPC:PS liposomes was only effective at pH < 5.5. We then designed a fluorescent ratiometric apoAI pH indicator labeling apoAI with FITC and Alexa647, and validated it in solution assays showing that the FITC/Alexa647 ratio decreased as the pH decreased. Using flow cytometry in both RAW264.7 and BHK cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37 o C led to a large acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21 o C or with an inhibitor of endocytosis, where the vast majority of apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V 1 domain and an embedded V 0 domain. We observed that bafilomycin, the V-ATPase inhibitor, dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI, and also eliminated the pH shift of apoAI on plasma membrane. We showed V-ATPase was expressed on the cell surface by cell surface biotinylation. Although the total level of V 0 A1 subunit didn’t change in ABCA1 expressing cells, ABCA1 induced the cell surface level of the V 0 A1 subunit. Conclusion: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which may promote apoAI unfolding and lipidation.

Post-translational palmitoylation and glycosylation of Wnt-5a are necessary for its signalling

Wnt-5a is a representative ligand that activates a β-catenin-independent pathway in Wnt signalling. In the present paper, the roles of the post-translational modifications in the actions of Wnt-5a were investigated. We found that Wnt-5a is modified with palmitate at Cys104 and glycans at Asn114, Asn120, Asn311 and Asn325. The palmitoylation was not essential for the secretion of Wnt-5a, but was necessary for its ability to suppress Wnt-3a-dependent T-cell factor transcriptional activity and to stimulate cell migration. Wnt-5a activated focal adhesion kinase and this activation also required palmitoylation. Wild-type Wnt-5a induced the internalization of Fz (Frizzled) 5, but a Wnt-5a mutant that lacks the palmitoylation site did not. Furthermore, the binding of Wnt-5a to the extracellular domain of Fz5 required palmitoylation of Wnt-5a. These results indicate that palmitoylation of Wnt-5a is important for the triggering of signalling at the cell surface level and, therefore, that the lipid-unmodified form of Wnt-5a cannot activate intracellular signal cascades. In contrast, glycosylation was necessary for the secretion of Wnt-5a, but not essential for the actions of Wnt-5a. Thus the post-translational palmitoylation and glycosylation of Wnt-5a are important for the actions and secretion of Wnt-5a.