RESUMO
S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7-24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington's disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs.
Assuntos
Aciltransferases/genética , Motivos de Aminoácidos/genética , Repetição de Anquirina/genética , Dedos de Zinco/genética , Acilação , Aciltransferases/classificação , Aciltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Western Blotting , Bovinos , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Proteína Huntingtina , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Filogenia , Ligação Proteica , Ratos , Homologia de Sequência de Aminoácidos , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are 24 ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzyme-substrate specificity and unravelling the importance of this enzyme family for cellular physiology and pathology.
Assuntos
Acilação/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Humanos , Família Multigênica/genética , Especificidade por SubstratoRESUMO
S-acylation, the attachment of fatty acids onto cysteine residues, regulates protein trafficking and function and is mediated by a family of zDHHC enzymes. The S-acylation of peripheral membrane proteins has been proposed to occur at the Golgi, catalyzed by an S-acylation machinery that displays little substrate specificity. To advance understanding of how S-acylation of peripheral membrane proteins is handled by Golgi zDHHC enzymes, we investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins-synaptosomal-associated protein 25 (SNAP25) and cysteine-string protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The ankyrin-repeat domains of zDHHC17 and zDHHC13 mediated strong and selective interactions with SNAP25/CSP, whereas binding of zDHHC3 and zDHHC7 to these proteins was barely detectable. Despite this, zDHHC3/zDHHC7 could S-acylate SNAP25/CSP more efficiently than zDHHC17, whereas zDHHC13 lacked S-acylation activity toward these proteins. Overall the results of this study support a model in which dynamic intracellular localization of peripheral membrane proteins is achieved by highly selective recruitment by a subset of zDHHC enzymes at the Golgi, combined with highly efficient S-acylation by other Golgi zDHHC enzymes.