New functional motif in Hox domain-containing ceramide Synthases - Identification of a novel region flanking the Hox and TLC domains essential for activity
New functional motif in Hox domain-containing ceramide Synthases - Identification of a novel region flanking the Hox and TLC domains essential for activity
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DOI：
10.1074/jbc.m703487200
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发表时间：
2007-09-14
影响因子：
4.8
通讯作者：
Futerman, Anthony H.
中科院分区：
文献类型：
--
作者：
Mesika, Adi;Ben-Dor, Shifra;Futerman, Anthony H.
神经酰胺是由一系列神经酰胺合成酶(CERs)在哺乳动物中合成的，每个CERs都使用一组相对有限的脂肪酰基-COA来进行狮身人面像长链碱基的N-酰化(Pewzner-Jung，Y.，Ben-Dor，S.和Futerman，A.H.(2006)J.Biol)。化学。281、25001-25005)。CERs有两个功能结构域，即bar ram-(L)下的(T)和bar LN8(TLC)结构域下的bar ag-(C)和同源框(Hox)结构域，该结构域存在于除CerS1以外的所有哺乳动物CER中。我们现在证明了大多数HOX结构域是CERs活性所不需要的，因为它在CerS5中的删除不会影响活性。随后，我们定义了一个高度保守的新基序，由12个氨基酸残基组成，位于Hox和TLC结构域的两侧，但不是TLC结构域的一部分，TLC结构域是CerS5和CerS6活性所必需的。该结构域上的两个带正电荷的残基，其中一个在所有可能的CER中都是保守的，对活性是必不可少的，因为任何一个(CerS5中的Lys-134和Lys-140)的定点突变都会导致类似50%的活性丧失，而这两个残基的突变都会导致活性的完全丧失。由于该区域在物种间是保守的，我们认为它在CERs活动中扮演着以前未知的重要角色，可以作为一个新的基序来定义包含Hox结构域的CERs。
Ceramide is synthesized in mammals by a family of ceramide synthases (CerS) each of which uses a relatively restricted set of fatty acyl-CoAs for N-acylation of the sphingoid long chain base (Pewzner-Jung, Y., Ben-Dor, S., and Futerman, A. H. ( 2006) J. Biol. Chem. 281, 25001-25005). CerS are characterized by two functional domains, the (T) under bar ram-(L) under bar ag-(C) under bar LN8 (TLC) domain and the homeobox (Hox) domain, which is found in all mammalian CerS except CerS1. We now demonstrate that the majority of the Hox domain is not required for CerS activity since its deletion in CerS5 does not affect activity. Subsequently, we define a highly conserved new motif of 12 amino acid residues that flanks the Hox and TLC domains but is not part of the TLC domain, which is essential for CerS5 and CerS6 activity. Two positively charged residues in this domain, one of which is conserved in all putative CerS in all organisms, are essential for activity since site-directed mutagenesis of either (Lys-134 and Lys-140 in CerS5) results in an similar to 50% loss of activity, whereas mutation of both leads to a complete loss of activity. Because this region is conserved across species, we propose that it plays a previously unidentified and essential role in CerS activity and can be used as a new motif to define Hox domain-containing CerS.