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CIITA is 1130 amino acid protein and is dynamically regulated through an intricate series of posttranslational modifications (PTMs) .
We hypothesized pCAF is playing a novel role as ubiquitin E3 ligase for CIITA in addition to its traditional role as HAT.
Cells were transfected using GeneJuice (Merck Millipore, Darmstadt, Germany) as indicated with 5 [micro]g of Myc-CIITA, Flag-pCAF, Flag-K141R, K144R, K141/144R CIITA, HA-K48 Ub, K63 Ub, HA-mono Ub, or pCDNA control.
The CIITA in vitro ubiquitination assay was carried out in 150 [micro]L of reaction mixture containing 40 mM Tris-HCL (pH 7.5), 5mM Mg[Cl.sub.2], 2mM dithiothreitol, 1mM Creatine Phosphate, 2mM ATP, 400 ng of Recombinant GST-CIITA (substrate), 400 ng of GSTHdm2 (Boston Biochem), 400 ng Recombinant His-pCAF (E3 ligase candidate) (Proteinone Rockville, MD), 500 ng Flag ubiquitin (Boston Biochem, Boston, MA), 200 ng E1 activating enzyme, UBE1 (Boston Biochem), 200 ng E2 conjugating enzyme, and UbcH5b (Boston Biochem).
pCAFs E3 Ligase Domain Is Necessary for CIITA Transactivity.
Gross, "Toxoplasma gondii inhibits MHC class II expression in neural antigen-presenting cells by downregulating the class II transactivator CIITA," Journal of Neuroimmunology, vol.
These RFX members have been shown to interact with the class II transactivator (CIITA) [37, 38], which is also an important element for HLA class I gene transactivation .
Therefore, CIITA, which is dependent on a functional SXY module, does not transactivate HLA-G, mainly because of the missing X2 and Y elements 25, 41, 42, 47, 48].
Steimle, "CIITA leucine-rich repeats control nuclear localization, in vivo recruitment to the major histocompatibility complex (MHC) class II enhanceosome, and MHC class II gene transactivation," Molecular and Cellular Biology, vol.
Strominger, "Specific complex formation between the type II bare lymphocyte syndromeassociated transactivators CIITA and RFX5," Proceedings of the National Academy of Sciences of the United States of America, vol.
1 Idcirco, hoc edicto singuli universique cognoscant pantapolis ad urbem Roman redeundi negotandique licentiam restituam, ut cura pervigli ubertas populo ministretur et in rebus suspectis a maiore multitudnie ciitas possit habitari, ita ut, si quis statuta pretia non servaverit, in eum pro qualitate facti vindicta procedat nec ob unius aut alterius delitum omne corpus liceat excludi.
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