Identification of a novel sequence motif recognised by the ankyrin-repeat domain of zDHHC17/13 S-acyl-transferases

S-acylation is a major post-translational modification affecting several cellular processes and being particularly important for neuronal functions. This modification is catalysed by a family of transmembrane S-acyl-transferases that contain a conserved zinc-finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7-24 distinct zDHHC enzymes, with 2 members also harbouring an ankyrin-repeat (AR) domain at their cytosolic N-terminus. 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 recognised by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localised zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; these are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington disease. Here, we report that zDHHC17 and zDHHC13 recognise 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 recognised by AR-containing zDHHCs

The zDHHC family of S-acyltransferases

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

Identification of key features required for efficient S-acylation and plasma membrane targeting of Sprouty-2

Sprouty-2 is an important regulator of growth factor signalling and a tumour suppressor protein. The defining feature of this protein is a cysteine-rich domain (CRD) that contains twenty-six cysteines and is modified by S-acylation. In this study, we show that the CRD of Sprouty-2 is differentially modified by S-acyltransferase enzymes. The high specificity/low activity zDHHC17 enzyme mediated restricted S-acylation of Sprouty-2, and cysteines-265/268 were identified as key targets of this enzyme. In contrast, the low specificity/high activity zDHHC3/zDHHC7 enzymes mediated more expansive modification of the Sprouty-2 CRD. Nevertheless, S-acylation by all enzymes enhanced Sprouty-2 expression, suggesting that S-acylation stabilises this protein. In addition, we identified two charged residues (aspartate-214 and lysine-223), present on opposite faces of a predicted alpha helix in the CRD, which are essential for S-acylation of Sprouty-2. Interestingly, mutations that perturbed S-acylation also led to a loss of plasma membrane localisation of Sprouty-2 in PC12 cells. This study provides insight into the mechanisms and outcomes of Sprouty-2 S-acylation, and highlights distinct patterns of S-acylation mediated by different classes of zDHHC enzymes

The Golgi S-acylation machinery comprises zDHHC enzymes with major differences in substrate affinity and S-acylation activity

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, catalysed 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 have investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins, SNAP25 and Cysteine-String Protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The Ankyrin-repeat (ANK) 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 whereby dynamic intracellular localisation 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

Energy Valorization of Fine Screenings from a Municipal Wastewater Treatment Plant

The aim of this paper was to evaluate the characteristics and the energy potential for the methane production of fine screenings collected from the primary stage of a municipal wastewater treatment plant, and assess the impact on the properties and the oxygen demand of the aqueous effluents downstream from the sieves. Commercial filter bags with sieve openings of 3000, 1250, 1000, and 300 μm were used for the collection of screenings following a measurement of their biochemical methane potential. It was revealed that solid fractions from the sieves with a large size presented a high net methane production capacity exceeding 900 mL/g VS, but the gas production rate was rather slow, requiring a long time to reach the final value. However, cumulative solid fractions containing particles with a size larger than 300 μm had a lower net methane production, about 700 mL/g VS, but with a faster rate, resulting in almost 80% of the total volume released in 30 days. Aqueous samples downstream from the sieves presented decreasing organic matter content by sieve size and reduced the requirements for aeration oxygen. The installation of fine sieves in existing municipal wastewater treatment plants, therefore, may be beneficial due to the enhancement of biogas production and a reduction in the oxygen consumption of the activated sludge process