Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine.

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.

Comparison of Physical and Biochemical Characterizations of SARS-CoV-2 Inactivated by Different Treatments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused huge social and economic distress. Given its rapid spread and the lack of specific treatment options, SARS-CoV-2 needs to be inactivated according to strict biosafety measures during laboratory diagnostics and vaccine development. The inactivation method for SARS-CoV-2 affects research related to the natural virus and its immune activity as an antigen in vaccines. In this study, we used size exclusion chromatography, western blotting, ELISA, an electron microscope, dynamic light scattering, circular dichroism, and surface plasmon resonance to evaluate the effects of four different chemical inactivation methods on the physical and biochemical characterization of SARS-CoV-2. Formaldehyde and ß-propiolactone (BPL) treatment can completely inactivate the virus and have no significant effects on the morphology of the virus. None of the four tested inactivation methods affected the secondary structure of the virus, including the α-helix, antiparallel ß-sheet, parallel ß-sheet, ß-turn, and random coil. However, formaldehyde and long-term BPL treatment (48 h) resulted in decreased viral S protein content and increased viral particle aggregation, respectively. The BPL treatment for 24 h can completely inactivate SARS-CoV-2 with the maximum retention of the morphology, physical properties, and the biochemical properties of the potential antigens of the virus. In summary, we have established a characterization system for the comprehensive evaluation of virus inactivation technology, which has important guiding significance for the development of vaccines against SARS-CoV-2 variants and research on natural SARS-CoV-2.

Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation.

S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.

Inactivation of SARS-CoV-2 by ß-propiolactone causes aggregation of viral particles and loss of antigenic potential.

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. ß-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly resulting in insufficient inactivation, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.

Production of inactivated gram-positive and gram-negative species with preserved cellular morphology and integrity.

There are many approaches available to produce inactive bacteria by termination of growth, each with a different efficacy, impact on cell integrity, and potential for application in standardized inactivation protocols. The aim of this study was to compare these approaches and develop a standardized protocol for generation of inactivated Gram-positive and Gram-negative bacteria, yielding cells that are metabolically dead with retained cellular integrity i.e., preserving the surface and limited leakage of intracellular proteins and DNA. These inactivated bacteria are required for various applications, for instance, when investigating receptor-triggered signaling or bacterial contact-dependent analysis of cell lines requiring long incubation times. We inactivated eight different bacterial strains of different species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA, bacterial membrane and intracellular DNA staining, and visualization by scanning electron microscopy. Based on these results, we discuss the bacterial inactivation methods, and their advantages and disadvantages to study host-microbe interactions with inactivated bacteria.

Anti-SARS-CoV-2 IgG antibody response among Indian COVID-19 patients using ß-propiolactone-inactivated, whole virus-based indirect ELISA.

BACKGROUND: Coronavirus disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome - coronavirus-2 (SARS-CoV-2) continues to affect many countries and large populations. Serologic assays for antibody detection aid patient diagnosis and seroepidemiologic investigations. METHODS: An indirect IgG ELISA was developed indigenously using ß-propiolactone (BPL) inactivated SARS-CoV-2. This assay was used for screening 200 healthy donor sera collected prior to COVID-19 emergence (2017-2019), 185 serum/plasma samples of confirmed COVID-19 patients (n = 137) and 57 samples of viral RNA positive asymptomatic contacts (n = 51). The IgG response was studied in relation to duration and severity of illness. RESULTS: The ELISA demonstrated 97 % specificity and IgG detection in >50 %, 80 %, 93.8 % and 100 % of the patients respectively during the first, second, third and fourth week of illness. IgG detection rate was higher in patients with severe disease (SD, 90.9 %) than those with mild disease (MD, 68.8 %) during the second week of illness (P = 0.027). IgG seropositivity among asymptomatic contacts was 64.7 %. IgG ELISA absorbance values were higher in SD than MD patients during the first 2 weeks of illness (P < 0.05). No significant difference was observed between the absorbance values of asymptomatic subjects and MD patients (P = 0.94). CONCLUSION: The BPL inactivated virus-based ELISA could detect IgG antibodies early and in a significant proportion of COVID-19 patients suggesting its potential utility as a supplement to the currently used viral RNA detection tests in patient diagnosis and contact screening algorithms.

The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and Cryo-ET.

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) resulted from the outbreak of SARS-CoV-2 in December 2019. Currently, multiple efforts are being made to rapidly develop vaccines and treatments to fight COVID-19. Current vaccine candidates use inactivated SARS-CoV-2 viruses; therefore, it is important to understand the architecture of inactivated SARS-CoV-2. We have genetically and structurally characterized ß-propiolactone-inactivated viruses from a propagated and purified clinical strain of SARS-CoV-2. We observed that the virus particles are roughly spherical or moderately pleiomorphic. Although a small fraction of prefusion spikes are found, most spikes appear nail shaped, thus resembling a postfusion state, where the S1 protein of the spike has disassociated from S2. Cryoelectron tomography and subtomogram averaging of these spikes yielded a density map that closely matches the overall structure of the SARS-CoV postfusion spike and its corresponding glycosylation site. Our findings have major implications for SARS-CoV-2 vaccine design, especially those using inactivated viruses.

Propagation, Inactivation, and Safety Testing of SARS-CoV-2.

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.

Evaluation of Different Stabilizers and Inactivating Compounds for the Enhancement of Vero Cell Rabies Vaccine Stability and Immunogenicity: In Vitro Study.

Inactivation of rabies virus is essential for rabies vaccine preparation where the inactivating compound that is currently recommended for rabies vaccine preparation is ß-propiolactone (ß-PL). This compound is considered better than phenol and formalin but it is expensive and potentially carcinogenic. Data revealed that Ascorbic acid (AA) with cupric ions could yield complete and irreversible inactivation of rabies virus without adversely affecting its antigenicity. Additionally, the results of testing the vaccine potency with the selected inactivating compounds were comparable (P<0.05), and ED50 was higher than the recommended World Health Organization (WHO) limits. The use of HemaGel (plasma substitute) for testing vaccine stabilization was compared with the currently used vaccine stabilizers (human albumin and lactose). HemaGel yielded better stability than the other tested stabilizers. Monitoring of cellular and humoral immune responses indicated that both the total IgG level against rabies vaccine and the IFN and IL5 levels obtained with the HemaGel-stabilized vaccines were higher than those obtained with human albumin- and lactose-stabilized vaccine candidates.

Beta-propiolactone inactivated bivalent bluetongue virus vaccine containing Montanide ISA-71VG adjuvant induces long-term immune response in sheep against serotypes 4 and 16 even after 3 years of controlled vaccine storage.

In this study, we developed and evaluated the beta-propiolactone inactivated bivalent bluetongue virus (BTV) serotypes 4 and 16 vaccine delivered with Montanide™ ISA-71VG adjuvant. The safety, stability and immunological profile of the fresh and after three years of long-term storage of the vaccine formulation was analyzed. We observed after long-term storage that the vaccine emulsion was stable as indicated by unchanged pH and viscosity. The stored vaccine formulation induced virus neutralizing antibodies (VNA) in sheep against both the bluetongue virus serotypes at 7-10 day post-vaccination (dpv). VNA titers reached the peak by 60 dpv and detectable during the entire study period. Antibodies against bluetongue virus structural protein VP7 were detected by ELISA in all BTV vaccinated experimental animal groups. Partial clinical protection was observed in vaccinates against challenge virulent BTV-4 and BTV-16 serotypes by 10 dpv, while complete protection was observed at 14 dpv. The levels of viremia was decreased in challenged sheep by 10 dpv while the viremia was undetectable by 14 dpv. In summary, our newly formulated bivalent BTV (BTV-4 and BTV-16) vaccine delivered with Montanide™ ISA-71VG adjuvant was found safe and stable for over three years and induced protective response in sheep.

A Fluorescent Probe with Improved Water Solubility Permits the Analysis of Protein S-Depalmitoylation Activity in Live Cells.

S-Palmitoylation is an abundant lipid post-translational modification that is dynamically installed on and removed from target proteins to regulate their activity and cellular localization. A dearth of tools for studying the activities and regulation of protein S-depalmitoylases, thioesterase "erasers" of protein cysteine S-palmitoylation, has contributed to an incomplete understanding of the role of dynamic S-palmitoylation in regulating proteome lipidation. Recently, we developed "depalmitoylation probes" (DPPs), small molecule probes that become fluorescent upon S-depalmitoylase enzymatic activity. To be suitable for application in live cells, the first-generation DPPs relied on a shorter lipid substrate (C8 vs naturally occurring C16), which enhanced solubility and cell permeability. However, the use of an unnatural lipid substrate on the probes potentially limits the utility of the approach. Herein, we present a new member of the DPP family, DPP-5, which features an anionic carboxylate functional group that increases the probe water solubility. The enhanced water solubility of DPP-5 permits the use of a natural, palmitoylated substrate (C16), rather than a surrogate lipid. We show that DPP-5 is capable of monitoring endogenous S-depalmitoylases in live mammalian cells and that it can reveal changes in S-depalmitoylation levels due to lipid stress. DPP-5 should prove to be a useful new tool for probing the regulation of proteome lipidation through dynamic S-depalmitoylation.

Beta-Propiolactone Inactivation of Coxsackievirus A16 Induces Structural Alteration and Surface Modification of Viral Capsids.

Beta-propiolactone (BPL) is an inactivating agent that is widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we present cryo-electron microscopy (cryo-EM) structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids at resolutions of 3.9 Å and 6.5 Å, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N terminus of VP1 capsid protein, and the absence of pocket factor. However, major neutralizing epitopes are very well preserved on these particles. Further biochemical analyses revealed that BPL treatment impairs the abilities of CVA16 particles to bind to the attachment receptor heparan sulfate and to a conformation-dependent monoclonal antibody in a BPL dose-dependent manner, indicating that BPL is able to modify surface-exposed amino acid residues. Taken together, our results demonstrate that BPL treatment may induce alteration of the overall structure and surface properties of a nonenveloped viral capsid, thus revealing a novel mode of action of BPL.IMPORTANCE Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that the BPL concentration affects the binding of inactivated CVA16 particles to their receptor/antibody. Thus, BPL treatment can alter the overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.

Effects of Three Types of Inactivation Agents on the Antibody Response and Immune Protection of Inactivated IHNV Vaccine in Rainbow Trout.

Infectious hematopoietic necrosis virus (IHNV) infects salmonid fish, resulting in high mortality and serious economic losses to salmonid aquaculture. Therefore, an effective IHNV vaccine is urgently needed. To select an inactivation agent for the preparation of an effective IHNV vaccine, rainbow trout were immunized with mineral oil emulsions of IHNV vaccines inactivated by formaldehyde, binary ethylenimine (BEI), or ß-propiolactone (BPL). The fish were challenged 8 weeks after vaccination, and their IgM antibody response and relative percent survival (RPS) were evaluated. The results show that formaldehyde, BEI, and BPL abolished IHNV HLJ-09 infectivity within 24, 48, and 24 h at final concentrations of 0.2%, 0.02%, and 0.01%, respectively. The mean levels of specific IgM, both in serum and mucus (collected from the skin surface and gills), for the three immunized groups (from high to low) ranked as follows: the BPL group, BEI group, and formaldehyde group. From weeks 5 to 9, the mean log2 serum titers of IgM in the BPL group were significantly higher compared with those of the other groups (p < 0.05) during the 9 weeks of observation after vaccination (immunized at weeks 0 and6). Mucus OD490 values of the BPL group were significantly higher compared with those of the other groups (p < 0.05) when reaching their peak at weeks 5 and 8, but the difference between the formaldehyde and BEI groups was not significant (p > 0.05). The BPL-inactivated whole-virus vaccine had the greatest protective effect on the rainbow trout after challenge by an intraperitoneal injection of live IHNV, with an RPS rate of 91.67%, which was significantly higher compared with the BEI (83.33%) and formaldehyde (79.17%) groups. These results indicate that the BPL-inactivated IHNV oil-adjuvant vaccine was more effective than the formaldehyde- or BEI-inactivated vaccines. The results of this study provide an important foundation for further studies on inactivated IHNV vaccines.

Analysis of the beta-propiolactone sensitivity and optimization of inactivation methods for human influenza H3N2 virus.

Beta-propiolactone (BPL) is used as an inactivating reagent for influenza virus in a number of countries. However, the treatment of viruses with BPL occasionally results in a decrease in the hemagglutinin (HA) titer, which complicates vaccine development. In the present study, we examined the biological and biochemical characteristics of human H1N1 and H3N2 viruses treated with BPL, and developed an inactivation method for BPL-sensitive viruses. A significant decrease in HA titer was detected in the H3N2 viruses examined. The decrease in the pH of the virus fluid was not associated with the decreased HA titer, indicating that the decrease in HA titer for the H3N2 virus is the result of the direct effect of BPL. Excessive modification of M1 by BPL and loss of virion diameter were observed in 0.1% BPL-treated H3N2 virus. Taken together, these results suggest that the BPL sensitivity of H3N2 virus results from disruption of the virion. By contrast, the H3N2 virus was successfully inactivated by 0.02% BPL without a significant decrease in the HA titer or disruption of virion structure. Furthermore, we found that the 0.02% BPL in the virion preparation was hydrolyzed successfully by incubation at 37°C for 7h. Thus, mild treatment with a low concentration of BPL enabled us to inactivate the H3N2 virus.

Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells.

Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is not possible yet, modulators of NRAS posttranslational modifications have become an area of interest. Specifically, interfering with NRAS posttranslational palmitoylation/depalmitoylation cycle could disturb proper NRAS localization, and therefore decrease cell proliferation and downstream signaling. Here, we investigate the expression and function of NRAS depalmitoylating acyl protein thioesterases 1 and 2 (APT-1, APT-2) in a panel of NRAS mutant melanoma cells. First, we show that all melanoma cell lines examined express APT-1 and APT-2. Next, we show that siRNA mediated APT-1 and APT-2 knock down and that the specific APT-1 and -2 inhibitors ML348 and ML349 have no biologically significant effects in NRAS mutant melanoma cells. Finally, we test the dual APT-1 and APT-2 inhibitor palmostatin B and conclude that palmostatin B has effects on NRAS downstream signaling and cell viability in NRAS mutant melanoma cells, offering an interesting starting point for future studies.

Evaluation of different inactivation methods for high and low pathogenic avian influenza viruses in egg-fluids for antigen preparation.

In view of the emerging avian influenza (AI) viruses, it is important to study the susceptibility of AI viruses to inactivating agents for preparation of antigens and inactivated vaccines. The available information on susceptibility of both the high and low pathogenic AI viruses to different inactivating agents is inadequate and ambiguous. It has been shown that different subtypes of influenza viruses require different physical and chemical conditions for inactivation of infectivity. The present study was undertaken to evaluate the use of beta-propiolactone (BPL), formalin and ether for inactivation and its impact on antigenicity of AI viruses. A total of nine high and low pathogenic AI viruses belonging to four influenza A subtypes were included in the study. The H5N1 viruses were from the clades 2.2, 2.3.2.1 and 2.3.4. The H9N2 virus included in the study was of the G1 genotype, while the H11N1 and H4N6 viruses were from the Eurasian lineage. The viruses were treated with BPL, formalin and with ether. The confirmation of virus inactivation was performed by two serial passages of inactivated viruses in embryonated chicken eggs. The infectivity of all tested AI viruses was eliminated using 0.1% BPL and 0.1% formalin. Ether eliminated infectivity of all tested low pathogenic AI viruses; however, ether with 0.2% or 0.5% Tween-20 was required for inactivation of the highly pathogenic AI H5N1 viruses. Treatment with BPL, ether and formalin retained virus hemagglutination (HA) titers. Interestingly ether treatment resulted in significant rise in HA titers (P<0.05) of all tested AI viruses. This data demonstrated the utility of BPL, formalin and ether for the inactivation of infectivity of AI viruses used in the study for the preparation of inactivated virus antigens for research and diagnosis of AI.

An inactivated yellow fever 17DD vaccine cultivated in Vero cell cultures.

Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with ß-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 µg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine.

Modulating effects of acyl-CoA synthetase 5-derived mitochondrial Wnt2B palmitoylation on intestinal Wnt activity.

AIM: To investigate the role of acyl-CoA synthetase 5 (ACSL5) activity in Wnt signaling in intestinal surface epithelia. METHODS: Several cell lines were used to investigate the ACSL5-dependent expression and synthesis of Wnt2B, a mitochondrially expressed protein of the Wnt signaling family. Wnt activity was functionally assessed with a luciferase reporter assay. ACSL5-related biochemical Wnt2B modifications were investigated with a modified acyl-exchange assay. The findings from the cell culture models were verified using an Apc(min/+) mouse model as well as normal and neoplastic diseased human intestinal tissues. RESULTS: In the presence of ACSL5, Wnt2B was unable to translocate into the nucleus and was enriched in mitochondria, which was paralleled by a significant decrease in Wnt activity. ACSL5-dependent S-palmitoylation of Wnt2B was identified as a molecular reason for mitochondrial Wnt2B accumulation. In cell culture systems, a strong relation of ACSL5 expression, Wnt2B palmitoylation, and degree of malignancy were found. Using normal mucosa, the association of ACSL5 and Wnt2B was seen, but in intestinal neoplasias the mechanism was only rudimentarily observed. CONCLUSION: ACSL5 mediates antiproliferative activities via Wnt2B palmitoylation with diminished Wnt activity. The molecular pathway is probably relevant for intestinal homeostasis, overwhelmed by other pathways in carcinogenesis.

Identification of palmitoyltransferase and thioesterase enzymes that control the subcellular localization of axon survival factor nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2).

The NAD-synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is a critical survival factor for axons and its constant supply from neuronal cell bodies into axons is required for axon survival in primary culture neurites and axon extension in vivo. Recently, we showed that palmitoylation is necessary to target NMNAT2 to post-Golgi vesicles, thereby influencing its protein turnover and axon protective capacity. Here we find that NMNAT2 is a substrate for cytosolic thioesterases APT1 and APT2 and that palmitoylation/depalmitoylation dynamics are on a time scale similar to its short half-life. Interestingly, however, depalmitoylation does not release NMNAT2 from membranes. The mechanism of palmitoylation-independent membrane attachment appears to be mediated by the same minimal domain required for palmitoylation itself. Furthermore, we identify several zDHHC palmitoyltransferases that influence NMNAT2 palmitoylation and subcellular localization, among which a role for zDHHC17 (HIP14) in neuronal NMNAT2 palmitoylation is best supported by our data. These findings shed light on the enzymatic regulation of NMNAT2 palmitoylation and highlight individual thioesterases and palmitoyltransferases as potential targets to modulate NMNAT2-dependent axon survival.

A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.