Insights Into Protein S-Palmitoylation in Synaptic Plasticity and Neurological Disorders: Potential and Limitations of Methods for Detection and Analysis

Abstract

S-palmitoylation (S-PALM) is a lipid modification that involves the linkage of a fatty acid chain to cysteine residues of the substrate protein. This common posttranslational modification (PTM) is unique among other lipid modifications because of its reversibility. Hence, like phosphorylation or ubiquitination, it can act as a switch that modulates various important physiological pathways within the cell. Numerous studies revealed that S-PALM plays a crucial role in protein trafficking and function throughout the nervous system. Notably, the dynamic turnover of palmitate on proteins at the synapse may provide a key mechanism for rapidly changing synaptic strength. Indeed, palmitate cycling on postsynaptic density-95 (PSD-95), the major postsynaptic density protein at excitatory synapses, regulates the number of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and thus affects synaptic transmission. Accumulating evidence suggests a relationship between impairments in S-PALM and severe neurological disorders. Therefore, determining the precise levels of S-PALM may be essential for understanding the ways in which this PTM is regulated in the brain and controls synaptic dynamics. Protein S-PALM can be characterized using metabolic labeling methods and biochemical tools. Both approaches are discussed herein in the context of specific methods and their advantages and disadvantages. This review clearly shows progress in the field, which has led to the development of new, more sensitive techniques that enable the detection of palmitoylated proteins and allow predictions of potential palmitate binding sites. Unfortunately, one significant limitation of these approaches continues to be the inability to use them in living cells.

Keywords: S-palmitoylation; biochemical methods; metabolic labeling; neurodegenerative diseases; synapse; synaptic plasticity.

FIGURE 1

Dynamic S-palmitoylation of proteins. S-Palmitoylation is a reversible and dynamic protein modification that is mediated by DHHC-palmitoyltransferases and acyl protein thioesterases that attach to and remove palmitate from cysteine residues of proteins, respectively.

FIGURE 2

Schematic overview of S-palmitoylated proteins that are crucial for excitatory synapse function. (A) PSD-95. Dendritically localized DHHC mediates activity-sensitive palmitoylation of PSD-95 and its synaptic clustering. Palmitoylated PSD-95 regulates glutamate receptors (NMDAR and AMPAR) recruitment at postsynaptic sites, thus affecting synaptic efficacy. Enhanced stimulation of glutamate receptors leads to depalmitoylation of PSD-95 and its dissociation from the PSD. As a result of PSD-95 depalmitoylation, the glutamate receptors internalize and the synapse strength decreases. (B) AMPAR. Palmitoylation of GluA1/A2 subunits on the transmembrane domain (TMD) accumulates AMPAR in the Golgi apparatus, whereas depalmitoylation triggers receptor trafficking to the cell surface. C-terminal palmitoylation of GluA1/A2 does not influence the glutamate-dependent steady-state surface expression of AMPAR. On the other hand, palmitoylated form of the receptor is more sensitive to AMPA- and NMDA-induced receptor internalization. (C) NMDAR. Palmitoylation of the NMDARs on a membrane-proximal region of the NR2A/2B subunits ensures proper surface delivery and increases the stability of NMDAR on the postsynaptic membrane via tyrosine phosphorylation. In contrast, palmitoylation of the NR2A/2B subunits in the middle of C-terminal region increases accumulation of the NMDAR in the Golgi apparatus and decreases receptor surface expression. In turn, depalmitoylation of the NR2A/2B subunits regulates release of NMDA receptors from the Golgi for surface delivery.

FIGURE 3

Schematic overview of S-palmitoylated proteins that are crucial for inhibitory synapse function. (A) GABAR. Palmitoylation of the γ2 subunit determines the stability and mobility of receptors in the postsynaptic membrane and is required for normal GABAergic inhibitory transmission. (B) Gephyrin. Gephyrin, palmitoylated on two cysteine residues forms stable clusters at the postsynaptic membrane. Thereby increasing the strength of GABAergic transmission. Stimulation of GABAergic transmission leads to gephyrin palmitoylation and membrane association, ultimately increasing the size of gephyrin clusters.

FIGURE 4

Schematic overview of S-palmitoylated signaling molecules that are important for synapse function. (A) LIM kinase-1. Palmitoylation targets LIMK1 to the spine membrane, where it is phosphorylated by membrane-bound activators such as Cdc42/PAK. Palmitoylated LIMK1 phosphorylates cofilin and thus regulates spine-specific actin polymerization and morphological plasticity. In contrast, non-palmitoylated LIMK1 remains inactive (B) GTPase Cdc42. Palmitoylated Cdc42 accumulates in dendritic filopodia modulating their maturation and is strongly concentrated in dendritic spines. In response to enhanced neuronal activity Cdc42 is depalmitoylated and dissociated from the spine. (C) Tyrosine-protein Fyn kinase. Palmitoylation of Fyn is required for its membrane localization and thus proper action. Palmitoylated Fyn phosphorylates various substrates important for the accurate synaptic functioning, e.g., PSD-95, NMDAR, GABAR.

FIGURE 5

Schematic overview of S-palmitoylated proteins and DHHC enzymes related to neurodegenerative diseases. (A) Alzheimer’s disease. S-PALM APP is enriched in lipid rafts where it is cleaved by BACE1. DHHC3/GODZ, DHHC4, DHHC7, and DHHC15 promote the S-PALM of BACE1, which may facilitate the amyloidogenic process. Additionally, DHHC12 is involved in the regulation of APP localization, trafficking, and metabolism. (B) Huntington’s disease. The S-PALM of HTT is crucial for protein localization and trafficking. PolyQ expansion in HTT leads to lower affinity of the protein for its specific DHHC17, resulting in a reduction of HTT palmitoylation. Ion-resistant mutant HTT exhibited an increase in toxicity and a propensity to form aggregates.

FIGURE 6

Schematic overview of S-palmitoylated proteins and DHHC enzymes related to neuropsychiatric diseases. (A) Schizophrenia. A reduction of the palmitoylation of many proteins, such as vesicular glutamate transporter 1 (VGLUT1), the small GTPase Ras, and myelin basic protein (MBP), has been observed in the dorsolateral prefrontal cortex of schizophrenia patients. DHHC8 is located in the microdeletion region of chromosome 22q11 and may increase the risk of schizophrenia. (B) Intellectual disability. DHHC15 and DHHC9 are implicated in X chromosome-linked ID (XLID). Genetic mutations of chromosome X led to the disruption of transcription of the dhhc15 gene in a female patient with severe non-syndromic XLID. Four mutations of the dhhc9 gene were identified in patients with XLID. (C) Major depressive disorder. The S-PALM of 5-HT receptors may be directly involved in receptor isomerization from an inactive to an active form and may represent a general feature that regulates constitutive receptor activity. S-PALM is a targeting signal that is responsible for the retention of 5-HT_1A receptors in lipid rafts, which represent scaffold platforms for receptor-mediated signaling.

FIGURE 7

Schematic representation of the metabolic labeling workflow. S-PALM proteins can be studied using biorthogonal reactions. The cells are first incubated with palmitate derivatives (e.g., Alk-C16 and 17-ODYA; metabolic incorporation). Fixed and permeabilized cells are then processed for CuAAC (click chemistry) with either a fluorophore or affinity (biotin) tag. At this step, total S-PALM can be visualized by microscopy. Otherwise, after cell lysis, the level of S-PALM can be evaluated by in-gel fluorescence, Western blot (WB), or mass spectrometry (MS). For the single-cell in situ imaging of S-PALM in target protein, cells are subjected to the proximity ligation assay (PLA) using specific antibodies.

FIGURE 8

Schematic overview of biochemical assays for S-PALM identification. (A) Acyl Biotin Exchange (ABE). (B) Palmitoyl Protein Identification and Site Characterization (PalmPISC). (C) Resin Assisted Capture (AcylRAC). (D) Acyl-PEG Exchange (APE). The upper panel shows the particular stages of each assay. TCEP, Tris(2-carboxyethyl)phosphine; NEM, N-ethylmaleimide; IAM, iodoacetamide; MMTS, methyl methanethiosulfonate; mPEG-Mal, methoxy-PEG-maleimide; Biotin-HPDP, N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide; MS/MS, tandem mass spectrometry.