S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects.

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord.

Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation.

Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via transmembrane AMPAR regulatory protein subunits) or NMDARs [via glutamate ionotropic receptor NMDA-type subunit 2B (GluN2B) subunits] only in its palmitoylated and extended conformation. In contrast, in its extended conformation, SAP97 associates with NMDARs, but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation, and its interactions are dynamic when associated with AMPARs and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains.

Palmitoylation of superoxide dismutase 1 (SOD1) is increased for familial amyotrophic lateral sclerosis-linked SOD1 mutants.

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.

Resolving the assembly state of herpes simplex virus during axon transport by live-cell imaging.

Neurotropic herpesviruses depend on long-distance axon transport for the initial establishment of latency in peripheral ganglia (retrograde transport) and for viral spread in axons to exposed body surfaces following reactivation (anterograde transport). Images of neurons infected with herpes simplex virus type 1 (HSV-1), acquired using electron microscopy, have led to a debate regarding why different types of viral structures are seen in axons and which of these particles are relevant to the axon transport process. In this study, we applied time-lapse fluorescence microscopy to image HSV-1 virion components actively translocating to distal axons in primary neurons and neuronal cell lines. Key to these findings, only a small fraction of viral particles were engaged in anterograde transport during the egress phase of infection at any given time. By selective analysis of the composition of the subpopulation of actively transporting capsids, a link between transport of fully assembled HSV-1 virions and the neuronal secretory pathway was identified. Last, we have evaluated the seemingly opposing findings made in previous studies of HSV-1 axon transport in fixed cells and demonstrate a limitation to assessing the composition of individual HSV-1 particles using antibody detection methods.

Two modes of herpesvirus trafficking in neurons: membrane acquisition directs motion.

Alphaherpesvirus infection of the mammalian nervous system is dependent upon the long-distance intracellular transport of viral particles in axons. How viral particles are effectively trafficked in axons to either sensory ganglia following initial infection or back out to peripheral sites of innervation following reactivation remains unknown. The mechanism of axonal transport has, in part, been obscured by contradictory findings regarding whether capsids are transported in axons in the absence of membrane components or as enveloped virions. By imaging actively translocated viral structural components in living peripheral neurons, we demonstrate that herpesviruses use two distinct pathways to move in axons. Following entry into cells, exposure of the capsid to the cytosol resulted in efficient retrograde transport to the neuronal cell body. In contrast, progeny virus particles moved in the anterograde direction following acquisition of virion envelope proteins and membrane lipids. Retrograde transport was effectively shut down in this membrane-bound state, allowing for efficient delivery of progeny viral particles to the distal axon. Notably, progeny viral particles that lacked a membrane were misdirected back to the cell body. These findings show that cytosolic capsids are trafficked to the neuronal cell body and that viral egress in axons occurs after capsids are enshrouded in a membrane envelope.

The Herpesvirus capsid surface protein, VP26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus.

Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.