Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder affecting motor neurons, resulting in progressive muscle weakness and death by respiratory failure. Protein and RNA ...aggregates are a hallmark of ALS pathology and are thought to contribute to ALS by impairing axonal transport. Mutations in several genes known to contribute to ALS result in deposition of their protein products as aggregates; these include TARDBP, C9ORF72, and SOD1. In motor neurons, this can disrupt transport of mitochondria to areas of metabolic need, resulting in damage to cells and can elicit a neuroinflammatory response leading to further neuronal damage. Recently, eight independent human genetics studies have uncovered a link between TANK-binding kinase 1 (TBK1) mutations and ALS. TBK1 belongs to the IKK-kinase family of kinases that are involved in innate immunity signaling pathways; specifically, TBK1 is an inducer of type-1 interferons. TBK1 also has a major role in autophagy and mitophagy, chiefly the phosphorylation of autophagy adaptors. Several other ALS genes are also involved in autophagy, including p62 and OPTN. TBK1 is required for efficient cargo recruitment in autophagy; mutations in TBK1 may result in impaired autophagy and contribute to the accumulation of protein aggregates and ALS pathology. In this review, we focus on the role of TBK1 in autophagy and the contributions of this process to the pathophysiology of ALS.
Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which ...cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.
► Quantitative proteomic strategy reveals dynamics of cell-cycle protein interactions ► Cyclins confer biochemical specificity to cyclin/cdk interaction networks ► Cyclin A phosphorylates Sld3/c15orf42 and Wee1 to promote S phase and mitosis ► Cyclins A and B interact sequentially with proteins in G2 phase and mitosis
Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution ...of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.
Asexual stage Plasmodium falciparum replicates and undergoes a tightly regulated developmental process in human erythrocytes. One mechanism involved in the regulation of this process is ...posttranslational modification (PTM) of parasite proteins. Palmitoylation is a PTM in which cysteine residues undergo a reversible lipid modification, which can regulate target proteins in diverse ways. Using complementary palmitoyl protein purification approaches and quantitative mass spectrometry, we examined protein palmitoylation in asexual-stage P. falciparum parasites and identified over 400 palmitoylated proteins, including those involved in cytoadherence, drug resistance, signaling, development, and invasion. Consistent with the prevalence of palmitoylated proteins, palmitoylation is essential for P. falciparum asexual development and influences erythrocyte invasion by directly regulating the stability of components of the actin-myosin invasion motor. Furthermore, P. falciparum uses palmitoylation in diverse ways, stably modifying some proteins while dynamically palmitoylating others. Palmitoylation therefore plays a central role in regulating P. falciparum blood stage development.
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► A global approach identified >400 palmitoylated proteins in Plasmodium falciparum ► Palmitoyl proteins are central to invasion and other virulence-associated processes ► Palmitoylation is required for completion of the P. falciparum asexual life cycle ► P. falciparum uses palmitoylation dynamically for diverse regulatory purposes
Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative ...palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level.
S‐acylation (palmitoylation) is the only fully reversible lipid modification of proteins; however, little is known about how protein S‐acyltransferases (PATs) that mediate it are regulated. DHHC5 is ...a PAT that is mainly localised at the plasma membrane with roles in synaptic plasticity, massive endocytosis and cancer cell growth/invasion. Here, we demonstrate that DHHC5 binds to and palmitoylates a novel accessory protein Golga7b. Palmitoylation of Golga7b prevents clathrin‐mediated endocytosis of DHHC5 and stabilises it at the plasma membrane. Proteomic analysis of the composition of DHHC5/Golga7b‐associated protein complexes reveals a striking enrichment in adhesion proteins, particularly components of desmosomes. We show that desmoglein‐2 and plakophilin‐3 are substrates of DHHC5 and that DHHC5 and Golga7b are required for localisation of desmoglein‐2 to the plasma membrane and for desmosomal patterning. Loss of DHHC5/Golga7b causes functional impairments in cell adhesion, suggesting these proteins have a wider role in cell adhesion beyond desmosome assembly. This work uncovers a novel mechanism of DHHC5 regulation by Golga7b and demonstrates a role for the DHHC5/Golga7b complex in the regulation of cell adhesion.
Synopsis
Cell membrane localized DHHC5 palmitoylates components of desmosomes and promotes cell adhesion.
DHHC5 palmitoylates an accessory protein Golga7b.
Palmitoylated Golga7b inhibits DHHC5 internalization, which leads to accumulation of DHHC5 at the cell membrane.
Proteomic analysis reveals that desmosome components Desmoglein‐2 and Plakophilin‐3 are DHHC5 substrates.
DHHC5/Golga7b are required for Desmoglein‐2 palmitoylation, membrane localization and desmosome assembly.
Cell membrane localized DHHC5 palmitoylates components of desmosomes and promotes cell adhesion.
The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are ...used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases.
Protein palmitoylation (S‐acylation) has emerged as an important player in a range of cellular processes, and as a result, the palmitoyl‐acyltransferase (PAT) enzymes which mediate this modification ...have entered into the spotlight. Palmitoyltransferase ZDHHC5 (ZDHHC5) is among the more unique members of the PAT family as it is mainly localised to the plasma membrane and contains an extended cytoplasmic domain with several regulatory features. ZDHHC5 plays a vital role in a wide range of processes in different cell types. In this review, we offer a summary of the functions of ZDHHC5 in synaptic plasticity, cardiac function, cell adhesion and fatty acid uptake, among other processes. We also explore recent work has revealed several mechanisms to control the activity, localisation and function of ZDHHC5.
Palmitoyltransferase ZDHHC5 (ZDHHC5) is a member of the palmitoyl‐acyltransferase family of enzymes that S‐acylate proteins to regulate protein‐membrane interactions. Here, we review the function of ZDHHC5 in a range of biological processes and explore recent work which has revealed novel mechanisms to control the activity, localisation and function of ZDHHC5.
Abstract
Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups ...from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here, we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and could potentially deposit histone octamer onto nucleosome-depleted regions of DNA.
We isolated the postsynaptic density from human neocortex (hPSD) and identified 1,461 proteins. hPSD mutations cause 133 neurological and psychiatric diseases and were enriched in cognitive, ...affective and motor phenotypes underpinned by sets of genes. Strong protein sequence conservation in mammalian lineages, particularly in hub proteins, indicates conserved function and organization in primate and rodent models. The hPSD is an important structure for nervous system disease and behavior.
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