Palmitoylation and membrane interactions of cysteine string protein

S-palmitoylation is the reversible post-translational attachment of the 16-carbon saturated fatty acid palmitate to the sulphydryl group of cysteine residues through a thioester linkage. The primary function of palmitoylation has often been regarded as simply a means to increase the hydrophobicity and membrane affinity of modified proteins, allowing them to become stably membrane-associated. However, recent evidence has demonstrated additional roles for palmitoylation in regulating protein trafficking, the membrane micro-localisation of proteins, protein stability, and protein-protein interactions. A family of twenty-three mammalian proteins containing a conserved DHHC cysteine-rich domain have recently been identified, and several of these proteins have been shown to palmitoylate specific substrate proteins. The DHHC family of palmitoyl transferases are polytopic membrane proteins containing 4-6 membrane-spanning domains that are localised to distinct intracellular membranes, including the endoplasmic reticulum, Golgi apparatus, endosomes, and the plasma membrane. Since palmitoyl transferases are integral membrane proteins, substrate proteins must contain additional membrane targeting signals that mediate membrane association prior to palmitate transfer. For many proteins, the mechanism that mediates initial membrane binding prior to palmitoylation is clear; for example, initial membrane binding can be mediated by transmembrane domains or other lipid modifications such as prenyl or myristoyl groups, which are added to proteins in the cytosol. However, a number of palmitoylated proteins lack any obvious membrane targeting motifs and it is unclear how this class of proteins associate with membranes to allow palmitoylation to occur. The vesicle-associated, exocytotic chaperone Cysteine String Protein (CSP) is an example of such a protein. CSP is extensively palmitoylated on a "string' of fourteen cysteine residues present within its signature cysteine string domain. Palmitoylation of CSP is essential for its intracellular sorting and function, and thus it is important to understand how this essential modification of CSP is regulated. In this study, a detailed mutagenesis approach has been employed to elucidate the mechanisms governing the initial membrane targeting of CSP, and the enzymes that palmitoylate CSP have been identified. As well as providing important information on CSP palmitoylation and membrane interactions, it is hoped that this analysis will also serve as a paradigm to understand the mechanisms by which other proteins become palmitoylated. As a first step to characterising CSP membrane interactions, a hydrophobic 31 amino acid domain of CSP was identified as the minimal membrane binding domain present within the protein. This domain includes the cysteine string domain, and indeed cysteine residues within this domain are proposed to play an essential role in membrane interaction prior to palmitoylation. Membrane association of the minimal membrane binding domain is not sufficient to trigger palmitoylation, which requires additional residues downstream of the cysteine string domain. Intriguingly one role of these downstream residues in CSP appears to be to weaken membrane affinity and indeed, in contrast to the minimal membrane binding domain, full-length CSP was cytosolic in the absence of palmitoylation. The family of 23 DHHC proteins were screened for activity against CSP, showing that palmitoylation is specifically enhanced by co-expression of the Golgi-localised palmitoyl transferases DHHC3, DHHC7, DHHC15 or DHHC17; co-expression of these enzymes is sufficient to promote the stable membrane attachment of CSP. CSP mutants with an increased membrane affinity were localised to the ER, and thus physically separated from the Golgi-localised partner DHHC proteins of CSP, offering an explanation of why these mutants are not palmitoylated. Interestingly, palmitoylation of an ER-localised mutant could be rescued by Brefeldin A (BFA) treatment, which promotes the mixing of ER and Golgi membranes, supporting the view that mutants with an increased membrane affinity are not palmitoylated because they associate with 'inappropriate' membranes. In addition, the palmitoylated mutant remained at the ER following BFA washout and did not traffic to more distal membrane compartments, suggesting that palmitoylation of CSP may have a specific requirement to take place at Golgi membranes to facilitate subsequent intracellular sorting. A model is proposed whereby CSP utilises a weak membrane affinity to "sample" intracellular membranes for DHHC protein content. As partner DHHC proteins of CSP are restricted to the Golgi, palmitoylation and stable membrane attachment only occurs at this intracellular compartment. Mutations that enhance initial membrane affinity prevent sampling and lead to accumulation of CSP on abundant cellular membranes such as the ER. As a palmitoylated CSP mutant did not traffic from the ER, the coupling of CSP palmitoylation to Golgi membranes may therefore be an important requirement for subsequent sorting. These findings suggest that membrane "sampling" through specialised protein domains might be a common mechanism employed by substrate proteins to locate their specific DHHC partner proteins

Psychometric Properties of the Family Caregiver Delirium Knowledge Questionnaire

A valid, reliable measure of family caregivers’ knowledge about delirium was not located in the literature; such an instrument is essential to assess learning needs and outcomes of education provided. The purpose of the current study was to (a) develop a family Caregiver Delirium Knowledge Questionnaire (CDKQ) based on the Symptom Interpretation Model; and (b) establish validity and reliability of the measure. The 19-item CDKQ was developed and administered to 164 family caregivers for community-dwelling older adults. Descriptive statistics were examined for all variables. Psychometric testing included confirmatory factor analysis, item-to-total correlations, and internal consistency reliability. A three-factor model provided the best fit for the data. The findings support initial validity and reliability of the CDKQ with family caregivers. Although the CDKQ was developed for use with family caregivers, it has potential for use with other caregivers, such as home health aides

Evidence, theory and context - using intervention mapping to develop a school-based intervention to prevent obesity in children

© 2011 Lloyd et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Only limited data are available on the development and feasibility piloting of school-based interventions to prevent and reduce obesity in children. Clear documentation of the rationale, process of development and content of such interventions is essential to enable other researchers to understand why interventions succeed or fail

The linker domain of the SNARE protein SNAP25 acts as a flexible molecular spacer that ensures efficient S-acylation

S-Acylation of the SNARE protein SNAP25 (synaptosomeassociated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat- binding motif (zDABM) in SNAP25 ( 112VVASQP 117), which is downstream of its S-acylated, cysteine-rich domain ( 85CGLCVCPC 92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the “mini-linker” region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25

The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells

The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility. To generate an improved understanding of zDHHC2 trafficking and how this might be regulated by neuronal activity, we searched for intramolecular signals that regulate enzyme localisation. Two signals were mapped to the C-terminal tail of zDHHC2: a non-canonical dileucine motif [SxxxLL] and a downstream NP motif. Mutation of these signals enhanced plasma membrane accumulation of zDHHC2 in both neuroendocrine PC12 cells and rat hippocampal neurons, consistent with reduced endocytic retrieval. Furthermore, mutation of these signals also increased accumulation of the enzyme in neurites. Interestingly, several threonine and serine residues are adjacent to these sorting motifs and analysis of phospho-mimetic mutants highlighted a potential role for phosphorylation in regulating the efficacy of these signals. This study offers new molecular insight into the signals that determine zDHHC2 localisation and highlights a potential mechanism to regulate these trafficking signals

Substrate selectivity in the zDHHC family of S-acyltransferases

S-acylation is a reversible lipid modification occurring on cysteine residues mediated by a family of membrane-bound 'zDHHC' enzymes. S-acylation predominantly results in anchoring of soluble proteins to membrane compartments or in the trafficking of membrane proteins to different compartments. Recent work has shown that although S-acylation of some proteins may involve very weak interactions with zDHHC enzymes, a pool of zDHHC enzymes exhibit strong and specific interactions with substrates, thereby recruiting them for S-acylation. For example, the ankyrin-repeat domains of zDHHC17 and zDHHC13 interact specifically with unstructured consensus sequences present in some proteins, thus contributing to substrate specificity of these enzymes. In addition to this new information on zDHHC enzyme protein substrate specificity, recent work has also identified marked differences in selectivity of zDHHC enzymes for acyl-CoA substrates and has started to unravel the underlying molecular basis for this lipid selectivity. This review will focus on the protein and acyl-CoA selectivity of zDHHC enzymes

A cluster of palmitoylated cysteines are essential for aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis

Autosomal-dominant adult-onset neuronal cero id lipofuscinosis (ANCL) is caused by mutation of the DNAJC5 gene encoding cysteine string protein alpha (CSP α ). The disease- causing mutations, which result in substituti on of leucine-115 with an arginine (L115R) or deletion of the neig hbouring leucine-116 ( Δ L116) in the cysteine-string domain cause CSP α to form high molecular weight SDS-resistant aggregates, which are also present in post- mortem brain tissue from patients. Formation and stability of these mutant aggregates is linked to palmitoylation of the cysteine-str ing domain, however the regions of the mutant proteins that drive aggregatio n have not been determined. The importance of specific residues in the cysteine-string domain was in vestigated, revealing that a central core of palmitoylated cysteines is essential for aggregation of ANCL CSP α mutants. Interestingly, palmitoylated monomers of ANCL CSP α mutants were shown to be short-lived compared with wild-type CSP α, suggesting that the mutants eith er have a faster rate of depalmitoylation or that they are consumed in a time-dependent manner into high molecular weight aggregates. These findings provide new insight into the features of CSP α that promote aggregation in the presence of L115R/ Δ L116 mutations and reveal a change in the lifetime of palmitoyla ted monomers of the mutant proteins

Needs of caregivers in heart failure management: A qualitative study

This is a freely-available open access publication. The final version of this paper has been published in Chronic Illness, March 2015 by SAGE Publications Ltd, All rights reserved. It is available via the DOI in this record.Objectives: To identify the needs of caregivers supporting a person with heart failure and to inform the development of a caregiver resource to be used as part of a home-based selfmanagement programme. Methods: A qualitative study informed by thematic analysis involving 26 caregivers in individual interviews or a focus group. Results: Three distinct aspects of caregiver support in heart failure management were identified. Firstly, caregivers identified needs about supporting management of heart failure including: coping with the variability of heart failure symptoms, what to do in an emergency, understanding and managing medicines, providing emotional support, promoting exercise and physical activity, providing personal care, living with a cardiac device and supporting depression management. Secondly, as they make the transition to becoming a caregiver, they need to develop skills to undertake difficult discussions about the role; communicate with health professionals; manage their own mental health, well-being and sleep; and manage home and work. Thirdly, caregivers require skills to engage social support, and voluntary and formal services while recognising that the longterm future is uncertain. Discussion: The identification of the needs of caregiver has been used to inform the development of a home-based heart failure intervention facilitated by a trained health care practitioner.NIHR (Programme Grants for Applied Research