WO2025166210A1 - Targeted depalmitoylation as a therapeutic strategy - Google Patents

Abstract

The present disclosure relates to engineered depalmitoylases. S-Palmitoylation is a reversible post-translational modification that tunes the localization, stability, and function of an impressive array of proteins including ion channels, G-proteins, and synaptic proteins. Altered protein palmitoylation is linked to various human diseases including cancers, neurodevelopmental and neurodegenerative diseases. Disclosed herein is an engineered chemogenetic depalmitoylase that manipulates the palmitoylation status of target proteins using a chemically inducible dimerization system. In some aspects, the engineered chemogenetic depalmitoylase is programed to allow selective depalmitoylation in specific organelles, of individual protein complexes, or triggered by cell-signaling events. Also disclosed is an engineered depalmitoylase that allows depalmitoylation of proteins in response to Erk phosphorylation. An optogenetic actuator to manipulate protein palmitoylation is also disclosed. The disclosed engineered depalmitoylases represent versatile and powerful methods for manipulating protein palmitoylation in live cells.

Description

TARGETED DEPALMITOYLATION AS A THERAPEUTIC STRATEGY

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0001] This invention was made with government support under NS 110672 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0002] The present disclosure relates to engineered depalmitoylases with high specificity and applications thereof.

BACKGROUND

[0003] S-palmitoylation or S-acylation has emerged as a biologically consequential post- translational modification (PTM) essential for fine-tuning protein assembly, localization, and function. At the molecular level, palmitoylation involves the attachment of a 16-carbon palmitic acid to cysteine residues via a thioester bond. Unlike other lipid modifications, palmitoylation is a reversible process and for many proteins, the dynamic acylation/deacylation cycle is crucial for maintaining their steady-state subcellular distributions. Global proteomic analysis has revealed a ‘palmitoylome’ consisting of over 5000 distinct proteins that are palmitoylated, including various ion channel subunits (e.g. CaVp2A, Kchip2, BK channels, and NCX), G-proteins (NRas, HRas, and Ga/q), scaffolding proteins (PSD95), and kinases (Lyn and Fyn). Therefore, palmitoylation is thought to impact a dizzying array of biological processes. Indeed, altered protein palmitoylation and/or mutations in enzymes that mediate palmitoylation are linked to diverse human diseases including cancers, viral infections, diabetes, irritable bowel syndrome, cardiac disease, neurodevelopmental and neurodegenerative diseases. Consequently, selective manipulation of protein palmitoylation is highly desired both to delineate its complex physiology and as potential new therapies.

[0004] The addition of long chain fatty acyl-CoAs to cysteine residues is mediated by the family of multi-pass zine-finger transmembrane palmitoyl acyl transferases known as zDHHC proteins, named after the conserved aspartate-histidine-histidine-cysteine residues in their catalytic domain. The removal of fatty acid groups is catalyzed by diverse serine hydrolases such as: acyl-protcin thioesterases (APT1-2 or Lyplal-2), palmitoyl protein thioesterases (PPT1), and a/ hydrolase domain-containing protein family (Abhdl7a-c). Importantly, the dynamic acylation/deacylation cycle is essential for orchestrating proper trafficking, subcellular localization, and function of various palmitoylated proteins. At the cellular level, both acylation and deacylation are highly regulated, with zDHHC acyltransferases and various depalmitoylases exhibiting distinct subcellular localization and differential expression in various tissues. Furthermore, although both classes of enzymes show some preference for modifying specific targets or target sites, multiple depalmitoylases often act on a specific protein. The prevailing strategy to manipulate protein palmitoylation involves small molecule inhibitors or knockdown of specific zDHHC or depalmitoylases. Specifically, zDHHC proteins are inhibited by 2-bromopalmitate (2BP), tunicamycin, and cerulenin; however, these compounds have minimal selectivity and can lead to cytotoxicity, limiting their practical utility. Similarly, Palmostatin B and M have been developed as broad-spectrum inhibitors of depalmitoylases. More recently, selective compounds that target APT1, APT2, and ABHD17 classes of enzymes have been developed. Even still, as depalmitoylases are relatively promiscuous targeting many proteins across different tissues, these approaches likely evoke unintended consequences.

[0005] Despite these advances, several key challenges persist. First, to ensure proper subcellular trafficking of palmitoylated proteins, dynamic acylation and deacylation is thought to be precisely orchestrated by distinct zDHHCs and depalmitoylases that are differentially localized in various subcellular locations throughout the secretory pathway. Therefore, manipulating protein palmitoylation with subcellular precision would be highly advantageous. To this end, imaging approaches have been devised to both monitor the subcellular distribution of palmitoylated proteins and to assess the activity of depalmitoylases. Yet, there are limited strategies to preferentially depalmitoylate proteins from specific subcellular locations. Second, from a therapeutic perspective, selective manipulation of palmitoylation of specific proteins is essential to avoid off-target effects and cytotoxicity. However, given the relative promiscuity of zDHHCs and depalmitoylases, engineering target specificity has remained challenging. Third, multiple depalmitoylases are often co-expressed in particular cell types. Whether these proteins support redundant functions or evoke distinct physiological effects remains largely unknown. These gaps have hindered an in-depth understanding of complex mechanisms by which palmitoylation tunes protein function in physiology and for development of targeted therapeutics.

SUMMARY OF THE DISCLOSURE

[0006] Disclosed herein are modified depalmitoylases and their methods of production and use. In some embodiments, the modified depalmitoylase is modified from an enzyme in the Abhdl7 family. In certain embodiments, the depalmitoylase in the modified depalmitoylase is Abhdl7C.

[0007] In one aspect, the modified depalmitoylases comprises a depalmitoylase from a/ hydrolase domain-containing protein family, wherein the depalmitoylase includes an N-terminal region fragment and a catalytic domain fragment; a FK506 binding protein (FKBP); and a FKBP rapamycin binding domain (FRB). The FRB is linked to the N-terminal region fragment, and the FKBP is attached to the catalytic domain fragment. In some embodiments, the modified depalmitoylase further comprises a subcellular targeting sequence is attached to the N-terminal region fragment and directs the N-terminal region fragment to an intracellular target (for example, CavP2A, KChip2, Lyn, NRas, or Gaq). In some embodiments, the modified depalmitoylase further comprises a nanobody attached to the N-terminal region fragment. The nanobody specifically targets the N-terminal region fragment to bind to a target protein or target protein complex. In other embodiments, the modified depalmitoylase further comprises a nanobody attached to the catalytic domain fragment. The nanobody is specifically targets the catalytic domain fragment to bind to a target protein or target protein complex. The target protein or target protein complex may be at least one of CavP2A, KChip2, Lyn, NRas, and Gaq. These modified depalmitoylases may be used to depalmitoylate a target protein in a cell (for example, to manipulating neuronal synapses and excitability in a subject). The method comprises providing the modified depalmitoylase to a cell and providing rapamycin or a derivative thereof to the cell. For the method of manipulating neuronal synapses and excitability in a subject, the target protein of the modified depalmitoylase is CavP2A.

[0008] In another aspect, the modified depalmitoylase comprises a depalmitoylase from a/p hydrolase domain-containing protein family including an N-terminus and a C-terminus; a WW phospho-binding domain; and an Erk substrate with an FQFP ERK-docking site. The WW phospho-binding domain is linked to the N-terminus of the depalmitoylase. The Erk substrate with an FQFP ERK-docking site is linked to the C-terminus of the depalmitoylase. In some embodiments, the Erk substrate is from cdc24C. This modified depalmitoylase may be used to inhibit Ras activity in cells. The method comprises providing the modified depalmitoylase to the cells.

[0009] In yet another aspect, the modified depalmitoylase comprises a depalmitoylase from a/0 hydrolase domain-containing protein family, wherein the depalmitoylase includes an N-terminal region fragment and a catalytic domain fragment; and a protein pair that rapidly dimerize in the presence of a light signal. One member of the protein pair is attached to the N-terminal region fragment of the depalmitoylase, while the other member is attached to the catalytic domain fragment of the depalmitoylase. In some embodiments, the protein pair that rapidly dimerize in the presence of a light signal are eMagA and eMagB. In certain embodiments, eMagA is attached to the N-terminal region fragment of the depalmitoylase, and eMagB is attached to the C-terminal catalytic domain fragment of the depalmitoylase.

[0010] In one aspect of the method of producing modified depalmitoylases, the method is directed to producing a chemically activated depalmitoylase. The method comprises linking FKBP to a catalytic domain fragment from a depalmitoylase from a/p hydrolase domain-containing protein family; and linking FRB to a N-terminal region fragment from the depalmitoylase from a/p hydrolase domain-containing protein family. Providing rapamycin or a derivative thereof to the chemically activated depalmitoylase induces heterodimerization of the catalytic domain fragment and the N-terminal region fragment to create a catalytically active heterodimer. In some implementations, the method further comprises attaching a subcellular targeting sequence to the N-terminal region fragment. In some implementations, the method further comprises attaching a selective nanobody to the N-terminal region fragment and/or the catalytic domain fragment.

[0011] In another aspect of the method of producing modified depalmitoylases, the method comprises linking a WW phospho-binding domain to a depalmitoylase from a/p hydrolase domaincontaining protein family at its N-terminus; and linking an Erk substrate with an FQFP ERK- docking site to the depalmitoylase from a/p hydrolase domain-containing protein family at its C- terminus.

[0012] In yet another aspects, the method of producing a modified depalmitoylase comprises linking eMagA to a N-terminal region fragment from a depalmitoylase from a/p hydrolase domaincontaining protein family; and linking eMagB to a catalytic domain fragment from the depalmitoylase from a/p hydrolase domain-containing protein family. Providing blue light to the modified depalmitoyl ase induces heterodimerization of the catalytic domain fragment and the N- tcrminal region fragment to create a catalytically active hctcrodimcr.

[0013] The foregoing and other aspects, features, and advantages will be apparent from the DESCRIPTION and DRAWINGS, and from the CLAIMS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Implementations will hereinafter be described in conjunction with the appended and/or included DRAWINGS, where like designations denote like elements.

[0015] FIGS. 1A-1K show the design of functional validation of a chemogenetic depalmitoylase. FIG. 1A is a schematic show of the molecular architecture of Abhdl7 depalmitoylases. FIG. IB is a schematic of engineered chemogenetically-activated depalmitoylase that Abhdl7C is split with FRB attached to the N-terminus, and FKBP to C-terminus. FIG. 1C shows AlphaFold models of FacePalml7C with FKBP/FRB dimerization. FIG. ID shows confocal images depicting changes in CavPiA localization: (1) baseline (top), (2) Abhdl7C (middle), and (3) Abhdl7C S/A mutant. FIG. IE depicts confocal images showing time-dependent changes in colocalization of FacePalml7C NT (top) and CT (middle) and subsequent changes in Ca\ p2A localization (bottom). FIG. IF depicts a Diary plot showing changes in Pearson’s correlation showing colocalization of FacePalml7C NT and CT. Each dot, mean ± s.e.m. n = 12 cells from 2 independent transfections. FIG. 1G depicts a Diary plot that quantifies normalized cytosolic fluorescence of CavPiA with FacePalml7C (black) or with FacePalml7C S/A mutant (gray). Each dot, mean ± s.e.m. n = 35 (FacePalml7C) and 11 (S/A mutant) from 2 independent transfections. FIG. 1H is an Acyl-RAC assay that confirms FacePalml7C induced a reduction in palmitoylation of CavPiA-YFP. Top, palmitoylated fraction. Bottom, total input. HA, hydroxylamine. FIG. II is a bar graph showing relative density of palmitoylated CavP2A captured and eluted from resin compared to total input. Each bar, mean ± s.e.m. ****p < 0.0001 by unpaired t-test. FIG. 1J are Ba²⁺ current recordings of Cav2.2/CavP2A showing minimal inactivation with (blue) and without (black) rapamycin. With FacePalm, rapamycin boosts inactivation of Cav2.2/CavP2A. FIG. IK is a bar graph that summarizes changes in inactivation quantified as r800, the fraction of current remaining following 800ms of depolarization. Each bar, mean ± s.e.m. ****p < 0.0001 by one-way ANOVA followed by Tukcy’s multiple comparison’s test. Cav2.2/Cav02A without FaccPalml7C (baseline, n = 6; +rapa, n = 6). Cav2.2/CavP A with FacePalml7C (baseline, n = 10; 4-rapa, n = 12).

[0016] FIGS. 2A-2F illustrate the design and validation of FacePalm as a chemogenetic depalmitoylase. FIGS. 2A-2Ca are AlphaFold predictions of Abhdl7A structure (FIG. 2A), Abhdl7B structure (FIG. 2B), and Abhdl7C structure (FIG. 2C). NT contains the palmitoylated region that localized Abhdl7 enzymes to membrane compartments. The catalytic triad is shown in the dotted circular region. Abdhl7A-C differ in the variable region. The predictions are based on per-residue confidence score (pLDDT) with the darkest color corresponding to very high confidence regions. FIGS. 2D and 2E show the expression of YFP-tagged CavP2A, which confirms strong membrane localization with a PM marker (CellMask). Left, PM marker; Middle, CavP2A; Right, merge, e, Co-expression of Abhdl7c (top row), Abhdl7a (middle row), or Abhdl7b (bottom row) results in reduced membrane localization of CavP2A. FIG. 2F is a bar graph summarizing co-localization of CavP2A with a PM marker in the presence or absence of Abhdl7a- c. ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons test.

[0017] FIGS. 3A-3T depict a FacePalm showing a generalizable strategy for inducible protein depalmitoylation. FIG. 3A depicts an Acyl-RAC assay showing a reduction in palmitoylation of CavP2A-YFP with rapamycin activation of FacePalml7A. Top, palmitoylated fraction. Bottom, total input. HA, hydroxylamine. Marker, 100 kDa. FIG. 3B is a bar graph showing FacePalml7A induced changes in relative density of palmitoylated CavP2A captured and eluted from resin compared to total input. Each bar, mean ± s.e.m. from 3 independent trails. *p<0.05 by unpaired t-test. Top, palmitoylated fraction. Bottom, total input. FIG. 3C depicts confocal images showing time-dependent change in localization of CavP2A-YFP following activation of FacePalml7A by rapamycin. FIG. 3D depicts a Diary plot showing the average change in normalized cytosolic fluorescence (A _cyt/ o) following addition of rapamycin. Gray curve, the relationship with FacePalml7C reproduced from FIG. 1G. Each dot and error, mean ± s.e.m. (n = 20 cells from 3 independent transfections). FIGS. 3E and 3F depict the Acyl-RAC assay showing rapamycin activation of FacePalml7B reduced palmitoylation of CavP2A-YFP. Each bar, mean ± s.e.m. from 3 independent trails. Format as in FIGS. 3A and 3B. HA, hydroxylamine. Marker, 100 kDa. FIGS. 3G and 3H depict confocal images (FIG. 3G) and quantification (FIG. 3H) of time-dependent changes in AF_cyt/Fo of CavP2A-YFP following rapamycin activation of FacePalml7B. Each dot and error, mean ± s.e.m (n = 20). FIGS. 31 and 3J show that rapamycin activation of FacePalm 17C reduces palmitoylation of Kchip2-YFP detected with YFP antibody. Format is same as in FIGS. 3A and 3B. Each bar and error, mean ± s.e.m. from 3 independent trails. Markers, 50 and 70 kDa. FIGS. 3K and 3L show that the activation of FacePalml7C alters localization of Kchip2-YFP. Each dot, mean ± s.e.m. (n=20 cells from 3 transfections). Format is same as in FIGS. 3C and 3D. FIGS. 3M and 3N depict the Acyl-RAC assay showing rapamycin activation of FacePalml7C reduces palmitoylation of Lyn- YFP detected with YFP antibody. Format is same as in FIGS. 3 A and 3B. Each bar and error, mean ± s.e.m. from 3 independent trails. Marker, 70 kDa. FIGS. 30 and 3P show that FacePalml7C alters localization of Lyn- YFP. Each dot, mean ± s.e.m. (n=13 cells from 3 transfections). Format is same as in FIGS. 3C-3D. FIGS. 3Q-3R depict the Acyl-RAC assay showing FacePalml7C dependent reduction in palmitoylation of Venus-NRas detected with YFP antibody. Each bar and error, mean ± s.e.m. from 3 independent trails. Size marker, 50 kDa. FIGS. 3S and 3T depict confocal images and quantification showing altered localization of NRas following rapamycin activation of FacePalm 17C. Each dot and error, mean ± s.e.m. (n = 17 from 3 independent transfections).

[0018] FIGS. 4A-4L demonstrate organelle-targeted depalmitoylation by FacePalm. FIG. 4A is a schematic showing the secretory pathway and strategy for targeting FacePalm to specific organelles. FacePalniN is modified to contain targeting sequences to distinct organelles. Upon addition of rapamycin, FacePalmc containing the catalytic domain is recruited to the desired subcellular location, resulting in increased depalmitoylation of proteins from these regions. FIGS. 4B and 4C show that the activation of ER-targeted FacePalm results in minimal change in localization of CavP A-YFP. Confocal images are depicted in FIG. 4B. Diary plot shows normalized cytosolic fluorescence (FIG. 4C). Each dot and error, mean ± s.e.m from n = 24 cells. FIGS. 4D and 4E show that activation of Golgi-targeted FacePalm results in a modest increase cytosolic fluorescence. Format as in panels b-c. n = 25 cells. FIGS. 4F and 4G show that the activation of plasma-membrane localized FacePalm results in a strong increase in cytosolic fluoresce of CavP2A-YFP. Format is same as in FIGS. 4B and 4C. n = 22 cells. FIGS. 4H and 41 show that activation of early-endosome (Rab5) localized FacePalm also evokes a strong increase in cytosolic fluoresce of CavP2A-YFP. Format is same as in FIGS. 4B and 4C. n = 17 cells. FIGS. 4J and 4K show that activation of late-endosome localized FacePalm evoked a modest increase in cytosolic fluorescence. Format is same as in FIGS. 4B and 4C. n = 20 cells. FIG. 4L is a bar graph summarizing maximal change in cytosolic fluorescence in response to activation of FacePalm localized to distinct domains. Each bar, mean ± s.c.m. * p < 0.05, *** p < 0.005, and **** p < 0.0001 by one way ANOVA followed by Dunnett’s T3 multiple comparison’s test.

[0019] FIGS. 5A-5J show targeted depalmitoylation of specific protein complexes. FIG. 5A is a schematic showing the strategy for targeting FacePalm to individual protein complexes (FacePalm- it). FacePalmN is modified to include a nanobody that binds the desired target, here YFP. Activation of FacePalm results in selective depalmitoylation of YFP-tagged KChip2 but not Cer-tagged Cav 2A. FIG. 5B is a schematic showing design of a constitutively-active targeted depalmitoylases. Abhdl7cr containing its catalytic domain is tethered to a nanobody targeting the desired target. FIG. 5C shows confocal images of cells co-transfected with KChip2-YFP (green, left panels) and Cav 2A-Cer (red, right panels). FacePalm- it has a YFP-selective nanobody targeting Kchip2. Scale bar, 10 pm. FIG. 5D depicts Diary plot quantifying changes in cytosolic fluorescence of both KChip2-YFP (green dots and fit) and Cav02A-Cer (black dots and fit). Each dot, mean ± s.e.m. from n=23 cells (3 transfections). FIG. 5E depicts confocal images of cells cotransfected with KChip2-YFP (green, left panels) and CavP2A-mcherry (red, right panels) along with FacePalm-it that targets a CavP2A-selective nanobody. Scale bar, 10 pm. FIG. 5F depicts a Diary plot showing changes in normalized cytosolic fluorescence for both KChip2-YFP (green dots and fit) and CavP2A-mcherry (black dots and fit). Each dot, mean ± s.e.m. from n=28 cells (3 transfections). FIG. 5G shows both Kchip2 and C vP2A localized to the membrane at baseline. FIG. 5H shows that co-expression of Nb(YFP)-17c_cat increases cytosolic localization of Kchip2 but not CavP2A. FIG. 51 shows that co-expression of Nb(CavP2A)-17c_cat increases cytosolic localization of CavP2A but not Kchip2. FIG. 5J is bar graph quantifying ratio = Fcyt(CavP A)/F_Cyt(Kchip2), where F_cyt denotes the ratio of cytosolic fluorescence to total fluorescence in the cell. > 1 denotes higher level of CavP2A in cytosol thank Kchip2. Baseline (n = 70 cells); with Nb(YFP)-Abhdl7C_cat (n = 166 cells); with Nb(CavP2A)-17c_cat (n = 146 cells).

[0020] FIGS. 6A-6K show constitutive targeted depalmitoylation of proteins by engineered depalmitoylases. FIG. 6A is a schematic of the design strategy for a constitutively active depalmitoylase with target specificity. The catalytic domain of Abhdl7c was attached to a nanobody that binds YFP with high affinity (Nb(YFP)-17c_cat), thus recruiting the enzyme directly to the target protein and promoting its depalmitoylation. FIGS. 6B-6F show that co-expression of Nb(YFP)-17c_cat with Ca_vp_2A (FIG. 6B), Kchip2 (FIG. 6C), Lyn (FIG. 6D), NRas (FIG. 6E), and Ga_q (FIG. 6F) changes the membrane localization of these proteins. FIG. 6G is a schematic of the design of a constitutivcly-activc targeted dcpalmitoylascs. Top, Nb(YFP)-17c_Cat selectively targets YFP-tagged Kchip2. Bottom, Nb(CavP2A)-17c_cat selectively targets mCherry-tagged CavPiA. Confocal microscopy imaging show that Kchip2 and CavP2A are both localized to the membrane at baseline (FIG. 6H), and co-expression of Nb(YFP)-17c_cat increases cytosolic localization of Kchip2 but not CavP2A (FIG. 61), while co-expression of Nb(CavP2A)-17c_cat increases cytosolic localization of CavP2A but not Kchip2 (FIG. 6J). FIG. 6K is a bar graph quantifying ratio - F_Cyt(CavP2A)/ _Cyt(Kchip2), where F_cyt denotes the ratio of cytosolic fluorescence to total fluorescence in the cell. > 1 denotes higher level of CavP2A in cytosol than Kchip2. Baseline (n = 70 cells); with Nb(YFP)-Abhdl7C_cat (n = 166 cells); with Nb(CavP2A)-17c_cat (n = 146 cells). [0021] FIGS. 7A-7D depict engineering a depalmitoylation feedback loop for NRas. FIG. 7A is a schematic showing palmitoylation cycle of NRas. Increased NRas activity and downstream signaling results in activation of Erk. A depalmitoylase that is activated by Erk as a negative feedback loop to inhibit NRas in an activity dependent manner was engineered. FIG. 7B is a design strategy for Erk-activated depalmitoylase (dePalm-Er). The N-terminus of Abhdl7C was engineered with a WW motif. The C-terminus of Abhdl7C was engineered with an Erk substrate from cdc24C along with an FQFP ERK-docking site. Increased Erk-activity results in phosphorylation of Erk-substrate that then promotes dimerization of the N- and C-terminal regions of Abhdl7C, resulting in activation of the depalmitoylase. FIG. 7C show confocal images showing either wildtype (top) or Q[61]K mutant NRas (bottom) tagged with YFP in the presence of Golgi- targeted mCherry (golgin-mcherry). Left, at baseline both wild-type and mutant NRas exhibit strong membrane localization. Middle, co-expression of Abhdl7c increases golgi-localization of both wild-type and mutant NRas. Right, co-expression of dePalm-Er results in increased golgi- localization of Q[61]K mutant NRas but not wild-type. Scale bar, 10 pm. FIG. 7D is a bar graph summarizing Pearson’s correlation between NRas (yellow fluorescence from FIG. 7C) and golgi marker (red fluorescence from FIG. 7C). Each Bar, mean ± s.e.m. ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons test. For wild-type NRas, at baseline, n = 47 cells; with Abhdl7c, n = 51 cells; with dePalm-Er, n = 65 cells. For Q[61]K mutant NRas, at baseline, n = 48 cells; with Abhdl7c, n = 52 cells; with dePalm-Er, n = 72 cells.

[0022] FIGS. 8A-8D demonstrate that rapamycin alone has minimal effect on PSD95 clustering and AP firing properties in cultured hippocampal neurons. FIG. 8 A shows confocal images of hippocampal neurons with PSD95 immunostaining at baseline (left) and upon incubation with rapamycin for 4 h (right). Scale bar, 5 pm. FIG. 8B shows a cumulative histogram comparing the puncta density of PSD95 in the presence (red) and absence of rapamycin. Basal, n = 201 processes from 12 cells; +Rapa, n = 212 processes from 12 cells. FIG. 8C illustrates the exemplar traces from current clamp recordings showing action potentials evoked from hippocampal neurons (without FacePalm) in response to 4 pA current injection for 1 sec. Horizontal scale bar, 100 ms. Vertical scale bar, 20 mV. FIG. 8D shows AP firing rate in response to various current injections. Each dot, mean ± s.e.m. n = 9 cells (basal); n = 9 cells (+rapa).

[0023] FIGS. 9A-9P show bidirectional modulation of neuronal function by FacePalm variants. FIG. 9A is confocal images of hippocampal neurons transduced with FacePalml7A at baseline (left) and upon incubation with rapamycin for 4 h (right). PSD95 (green), mcherry marker for FacePalm expression (red), and DAPI (blue). Bottom, enlarged view. FIG. 9B is a cumulative histogram comparing changes in puncta density of PSD95 with FacePalml7A at baseline (black) or with rapamycin (red). Basal, n = 110 processes from 11 cells; +Rapa, n = 63 processes from 7 cells. **p=O.OO89 by Kolmogorov-Smirnov test. FIGS. 9C-9D show that activation of FacePalml7B reduces PSD95 puncta density in cultured hippocampal neurons. Format as in 9A- 9B. Basal, n = 127 processes from 11 cells; -i-Rapa, n = 103 processes from 12 cells. **p = 0.0032 by Kolmogorov-Smirnov test. FIGS. 9E-9F show that activation of FacePalml7C also reduces PSD95 puncta density. Format as in 9A-9B. Basal, n = 97 processes from 9 cells; -i-Rapa, n = 99 processes from 11 cells. **p = 0.0032 by Kolmogorov-Smimov test. ****p < 0.0001 by Kolmogorov-Smirnov test. FIG. 9G depicts exemplar traces from current clamp recordings showing action potentials evoked from hippocampal neurons transduced with FacePalml7A in response to 400 pA current injection for 1 sec. Black trace, basal; Red trace, with rapamycin. FIG. 9H shows AP firing rate in response to various current injections. Each dot, mean ± s.e.m. n = 12 cells (basal); n = 17 cells (+rapa). FIGS. 9I-9J show that activation of FacePalml7B results in increased AP firing. Exemplar traces (FIG. 91) and quantification of AP firing rate (FIG. 91). Each dot, mean ± s.e.m. n = 8 cells (basal); n = 10 cells (+rapa). *p < 0.05 by multiple t-tests. FIGS. 9K- 9L show that activation of FacePalmUC results in decreased AP firing. Exemplar traces (FIG. 9K) and quantification of AP firing rate (FIG. 9L). Each dot, mean ± s.e.m. n = 12 cells (basal); n = 9 cells (+rapa). *p < 0.05, **p < 0.01 by multiple t-tests. FIG. 9M depicts confocal images showing hippocampal neurons transduced with PSD95 -targeted FacePalm-it at baseline (left) and upon incubation with rapamycin for 4 h (right). FIG. 9N is a cumulative histogram comparing changes in the puncta density of PSD95 with FaccPalm-it targeting PSD95 at baseline (black) or with rapamycin (red). Basal, n = 241 processes from 37 cells; +Rapa, n = 186 processes from 27 cells. ***p=0.002 by Kolmogorov-Smirnov test. FIGS. 9O-9P show that activation of FacePalm- it targeting PSD95 minimally perturbs AP firing rate. Exemplar traces (FIG. 90) and quantification of AP firing rate (FIG. 9P). Each dot, mean ± s.e.m. n = 9 cells (basal); n = 11 cells (+rapa) from 3 independent cultures.

[0024] FIGS. 10A-10M show further validation of altered CavP2A localization upon activation of FacePalm by rapamycin. Cav02A is localized to the plasma-membrane at baseline (FIG. 10A). Left, PM marker; middle, CavP2A; right, merge. Following rapamycin activation of FacePalm for either 60 mins (FIG. 10B) or 120 mins (FIG. IOC), CavP2A is increasingly localized to the cytoplasm. Format as in FIG. 10A. FIG. 10D is Diary plot showing time-dependent changes in Pearson’s correlation (r_mem) between CavP2A and PM marker. Following rapamycin activation, a strong reduction in r_mem was observed. Each dot and error, mean ± s.e.m. from n = 8 cells from two independent transfections. FIG. 10E is Diary plot showing change in normalized cytosolic fluorescence (AF_cyt/Fo). Each dot and error, mean ± s.e.m. from n = 10 cells from two independent transfections. FIG. 10F shows that FacePalm induced reduction in r_mem linearly and correlates with the increase in CavP2A cytosolic fluorescence (AF_Cyt/Fo). FIG. 10G shows time constant for change in CavP2A estimated by fitting r_mem (1) or AF_cyt/Fo (2). Each dot, mean ± s.e.m. from n = 10 cells. Rapamycin alone fails to alter membrane localization CavP2A-YFP in the absence of FacePalm (FIG. 10H). Disabling the catalytic activity of FacePalml7C by introducing the S/A mutation in the catalytic triad abolishes rapamycin induced changes in localization of CavP2A (FIG. 101). FIGS. 10J and 10K show that blocking FacePalml7C catalytic activity with Palmostatin B, a pharmacological antagonist of Abhdl7c, also blocks rapamycin induced changes in CavP2A cytosolic fluorescence. Confocal images (FIG. 10J), AF_cyt/Fo (FIG. 10K). FIGS. 10L and 10M show that rapamycin activation of FacePalml7C promotes cytosolic localization of CavP2A-YFP even upon blocking new protein synthesis with cycloheximide. Confocal images (FIG. 10L), Fcyt/Fo (FIG. 10M).

[0025] FIGS. 11 A- 11C show that an optogenetic actuator allows reversible manipulation of protein palmitoylation. FIG. HA is a schematic showing design of opto-depalm, an optogenetic actuator to manipulate protein palmitoylation. Abhdl7NT is attached to eMagA while Abhdl7cr is attached to eMagB. With blue light illumination, eMag A and eMagB dimerize resulting in reconstitution and activation of the Abhdl7 holo-cnzymc. In dark, cMagA and eMagB dissociate rendering the enzyme inactive. FIG. 1 IB depict confocal images showing time-dependent changes in localization of CavP2A-mCherry in the absence (top row) or presence (bottom row) of opto- depalm. Blue light illumination from 0 to 60 mins. Scale bar, 10 pm. FIG. 11C is a Diary plot quantifying normalized cytosolic fluorescence of CavP2A with (dark gray dots) or without (black dots) opto-depalm. Region shaded in light gray corresponds to time period with blue light illumination. Each dot, mean ± s.e.m. n = 8 (with opto-depalm) and 16 (no opto-depalm) cells from 2 independent transfections.

[0026] FIGS. 12A-12O is the in-depth analysis showing robust translocation of FacePalml7CcT to desired subcellular domain following rapamycin activation. FIG. 12A is images showing, rapamycin activation results in minimal localization of CavP2A in the ER with untargeted FacePalml7C. In each row: left, ER marker; middle, FacePalml7CcT tagged with mCherry; right, merge. Top row shows cells in the absence of rapamycin and bottom row, cells exposed to rapamycin. FIG. 12B is images showing rapamycin activation results in a strong colocalization of FacePalml7CcT with ER with ER-targeted FacePalml7C. Format is same as in FIG. 12A. FIG. 12C is a bar graph summarizing changes in colocalization of FacePalml7CcT with an ER marker, with the untargeted or organelle targeted FacePalm approach. FIGS. 12D-12F show that rapamycin induced changes colocalization of FacePalm 17CCT with a golgi marker with untargeted (FIG. 12D) or golgi-targeted (FIG. 12E) FacePalm approach. With the organelle-targeted approach, FacePalml7C evokes a strong increase in golgi-localization following rapamycin activation (FIG. 12F). FIGS. 12G-12I show rapamycin induced changes in colocalization of FacePalml7Ccr with a PM marker with either untargeted (FIG. 12G) or golgi-targeted (FIG. 12H) FacePalm approach. Even with the untargeted approach, a significant increase in PM localization of FacePalml7CcT in the presence of rapamycin was observed. With the targeted approach, a further increase in localization of FacePalmUCcr to the PM was observed(FIG. 121). FIGS. 12J-12L show rapamycin induced changes in colocalization of FacePalml7Ccr with an early endosome marker with either untargeted (FIG. 12J) or early endosome-targeted (FIG. 12K) FacePalm approach. With the organelle-targeted approach, FacePalml7C evokes a strong increase in endosome-localization following rapamycin activation (FIG. 12L). FIGS. 12M-12O show rapamycin induced changes in colocalization of FacePalml7CcT with a late endosome marker with either untargeted (FIG. 12M) or late endosome-targeted (FIG. 12N) FacePalm approach. A strong increase in late endosomelocalization is also observed in targeted FaccPalml7C (FIG. 120). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons test.

[0027] FIGS. 13A-13J show that disabling the catalytic activity of FacePalm prevents changes in PSD95 localization and AP firing properties. Catalytically inactive FacePalm 17A S/A mutant evokes minimal changes in PSD95 clustering in the presence or absence of rapamycin (FIGS. 13A and 13B). Confocal images (FIG. 13A) and population data shown as a cumulative distribution of puncta density (FIG. 13B). basal, n = 389 processes from 28 cell; 4-rapa, 420 processes from 38 cells. FIGS. 13C and 13D show that catalytically inactive FacePalml7B S/A mutant also evokes minimal changes in PSD95 clustering in the presence or absence of rapamycin. Format same as in FIGS. 13A-13B. basal, n = 378 from 29 cells; -i-rapa n = 451 from 39 cells. FIGS. 13E-13F show that catalytically inactive FacePalml7C S/A mutant also evokes minimal changes in PSD95 clustering in the presence or absence of rapamycin. Format same as in FIGS. 13A and 13B. basal, n = 392 processes from 33 cells; + rapa, n = 355 processes from 30 cells. FIGS. 13G and 13H show current clamp recordings (FIG. 13G) and population analysis of AP firing rate (FIG. 13H) confirming minimal changes in neuronal AP firing with catalytically inactive FacePalm 17B S/A mutant. Each dot and error, mean ± s.e.m. from n = 9 (basal) and n = 7 (+rapa) cells from 3 independent cultures. FIGS. 131 and 13J show a minimal change in current clamp recordings (FIG. 131) and AP firing (FIG. 13J) with FacePalml7C S/A mutant. Each dot and error, mean ± s.e.m. from n = 10 (basal) and n = 7 (+rapa) cells from 3 independent cultures.

[0028] FIGS. 14A and 14B show that Cav2.2 channels are palmitoylated in HEK cells and in neurons. HA, hydroxylamine.

[0029] FIG. 15 shows that depalmitoylation of Cav2.2 by Abhdl7c diminishes channel function. The left graph depicts baseline whole-cell peak current densities of Cav2.2 recombinantly expressed in HEK293 cells. The middle graph depicts depalmitoylating Cav2.2 by overexpression of Abhdl7c reduces peak current density. The right graph shows that co-expression of Cav2.2 with a catalytically inactive Abhdl7 (Abhdl7c S211 A) prevents reduction in Cav2.2 currents.

[0030] FIGS. 16A-16C show that palmitoylation of Cav2.2 upregulates channel activity. The single channel analysis shows exemplary traces at the top and ensemble average Po-voltage relation in the bottom graphs. These results suggest that palmitoylation enhances Cav2.2 function by boosting channel openings.

[0031] FIGS. 17A-17C show that depalmitoyation of endogenous Cav2.2 results in reduced Ca²⁺ currents. FIG. 17A depicts exemplar traces show baseline Cav2.2 currents isolated pharmacologically from dorsal root ganglion cells expression GFP (control virus). FIG. 17B depicts that expression of Abhdl7c inhibits Cav2.2 currents. FIG. 17C depicts population data showing exemplar whole cell current density with cells expressing either GFP or Abhdl7c.

[0032] FIGS. 18A-18C show that targeted depalmitoylation diminishes endogenous Cav2.2 in DRG neurons. Nanobody nb.F3 that targets the Cav2.2 complex via the auxiliary Cavp subunit was fused with the catalytic domain of Abhdl7c. FIG. 18A depicts exemplary Cav2.2 currents from pharmacologically isolated from DRG neurons expressing nb.F3-Abhdl7C_cat S211 A mutant. Coexpression of nb.F3-Abhdl7C_cat diminished endogenous Cav2.2 currents (FIG. 18B). Targeted depalmitoylases, nb.F3-Abhdl7C_cat, diminishes Cav2.2 whole cell current density compared to nb.F3-Abhdl7C_cat S/A mutant (FIG. 18C).

DETAILED DESCRIPTION

[0033] Detailed aspects and applications of the disclosure are described below in the drawings and detailed description of the disclosure. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0034] In the following description, and for the purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that the present disclosure may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices, and technologies to which the disclosed disclosures may be applied. The full scope of the disclosures is not limited to the examples that are described below. [0035] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

[0036] The word “exemplary,” “example,” or various forms thereof are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” or as an “example” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Furthermore, examples arc provided solely for purposes of clarity and understanding and are not meant to limit or restrict the disclosed subject matter or relevant portions of this disclosure in any manner. It is to be appreciated that a myriad of additional or alternate examples of varying scope could have been presented but have been omitted for purposes of brevity.

[0037] When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0038] As required, detailed embodiments of the present disclosure are included herein. It is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limits, but merely as a basis for teaching one skilled in the art to employ the present invention. The specific examples below will enable the disclosure to be better understood. However, they are given merely by way of guidance and do not imply any limitation. [0039] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a pail of this disclosure. It is to be understood that this disclosure is not limited to the specific materials, devices, methods, applications, conditions, or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed inventions. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0040] More specifically, this disclosure, its aspects and embodiments, are not limited to the specific material types, components, methods, or other examples disclosed herein. Many additional material types, components, methods, and procedures known in the art are contemplated for use with particular implementations from this disclosure. Accordingly, for example, although particular implementations are disclosed, such implementations and implementing components may comprise any components, models, types, materials, versions, quantities, and/or the like as is known in the art for such systems and implementing components, consistent with the intended operation.

[0041] Given the emerging role of palmitoylation in diverse human diseases, harnessing (de)palmitoylation as a therapeutic modality has garnered considerable interest. In this regard, selective inhibitors of depalmitoylases have been used in pathophysiological settings. For example, the APT1 inhibitor ML348 has been shown to reverse neuronal function and behavioral phenotype in Huntington disease models. Similarly, the recently developed Abhdl7 inhibitor ABD957 has been used to inhibit NRas signaling and growth of NRAS-mutant human acute myeloid leukemia cells.

[0042] The present disclosure relates depalmitoylating proteins. Specifically, a modified depalmitoylase from a/p hydrolase domain-containing protein family is disclosed. In some aspects, toolkit described herein utilizes a feedback loop based on ERK phosphorylation as a strategy for disrupting membrane association of overactive signaling proteins, for example Ras proteins. The strategy can also depalmitoylating proteins from specific subcellular locations.

[0043] Strategies to dynamically manipulate protein palmitoylation are also highly desired for both identifying physiological regulatory mechanisms and potential therapeutics. However, the relative promiscuity of (de)acylating enzymes has made this a challenging endeavor. Available strategies to preferentially depalmitoylate proteins from specific subcellular locations are limited. Conventionally, the functional consequence of protein palmitoylation is deduced via site-specific mutagenesis of palmitoylated cysteine residues or using pharmacological blockers such as 2-BP. However, site-specific mutagenesis is unable to resolve spatial- and time-dependent changes in protein palmitoylation and the pharmacological agents have minimal selectivity leading to cytotoxicity that limits their physiological and therapeutic utility. To address these limitations, a toolkit of chemogenetically-activated depalmitoylases with high specificity is described herein.

[0044] In one aspect, a chemically activated depalmitoylases (FacePalm) was developed by engineering a depalmitoylase from a/[3 hydrolase domain-containing protein family (for example, from the Abhdl7 family) to be chemically activated by the heterodimerization of FK506 binding protein (FKBP) and FKBP rapamycin binding domain (FRB). Specifically, the modified depalmitoylase is modified with a FK506 binding protein (FKB) and a FKBP rapamycin binding domain (FRB) at the depalmitoylase’s N-terminal region fragment and catalytic domain fragment. The FRB is linked to the N-tcrminal region fragment, and the FKB is attached to the catalytic domain fragment. The FRB-attached N-terminal region (also referenced herein as “FacePalmN”) and the FKB-attached catalytic domain (also referenced herein as “FacePalmc”) dimerize to form a catalytically active heterodimer, and this dimerization is inducible with rapamycin or derivatives thereof.

[0045] In certain embodiments, Abhdl7 enzyme is into two fragments: (1) FaccPalmx containing the Abhdl7 N-terminal region attached to FRB that serves as a membrane targeting moiety and (2) FacePalmc containing the Abhdl7 catalytic domain attached to FKBP. This strategy is generalizable across the Abhdl7 family and can be used to inducibly depalmitoylate various target proteins including ion channel subunits, kinases, and G-proteins. This approach is advantageous as it can be further engineered to enable depalmitoylation from specific subcellular locations or with high target selectivity. In particular embodiments, the modified depalmitoylase is based on Abhdl7C.

[0046] In some embodiments, FacePalm further comprises a subcellular targeting sequence. The subcellular targeting sequence is attached to the N-terminal region fragment and directs the N- terminal region fragment to an intracellular target. For example, FacePalmN is attached to the subcellular targeting sequence.

[0047] In some implementations, FacePalm further comprises a nanobody. In some aspects, the nanobody is attached to the N-terminal region fragment, and the nanobody is specifically bind the N-terminal region fragment to bind to a target protein or target protein complex. For example, FacePalmN is attached to a nanobody that binds a target with high affinity. In other aspects, the nanobody is attached to the catalytic domain fragment allowing the catalytic domain fragment to bind to a target protein or target protein complex.

[0048] In certain implementations, the intracellular target or the target protein or target protein complex comprises at least one member selected from the group consisting of: CaVp2A, KChip2, Lyn, NRas, and Gaq.

[0049] Upon rapamycin activation, the catalytic domain of Abhdl7 is then recruited to the desired protein complex, permitting selective depalmitoylation of this target without perturbing other palmitoylated proteins. For example, in experiments, it was possible to selectively depalmitoylate either CavP A or Kchip2 by switching nanobodies. A key advantage is that this overall approach can be generalized by developing selective nanobodies or binding proteins for a target of interest. Moreover, instead of chcmogcnctic activation, it is also possible to constitutively promote depalmitoylation of a target by directly attaching a selective nanobody to the catalytic domain of Abhdl7. This overall molecular design may form the basis for a distinct class of palmitoylation modulators with potential therapeutic utility.

[0050] As shown in the Examples, the modified depalmitoylase successfully manipulates palmitoylation of a diverse family of proteins including CavP2A, an obligatory subunit of Cavl/2 channels that supports sustained Ca²⁺ influx during pacemaking of substantia nigra dopaminergic neurons that contributes to the selective vulnerability of these neurons; KChip2, a regulator of Kv4 channels, implicated as a seizure susceptibility gene and in association with cardiac arrhythmias; Lyn, a Src family tyrosine kinase in various cellular- processes; NRas, a small G-protein and a commonly mutated oncogene in human cancer; and PSD95, a synaptic scaffolding protein. The results show the described modified depalmitoylases can be leveraged for (1) depalmitoylation of proteins from specific subcellular locations, (2) targeted depalmitoylation of individual protein complexes, and (3) inducible depalmitoylation in response to cell signaling events. This depalmitoylation platform represents a powerful strategy to tune protein palmitoylation with high precision and, in so doing, opens new frontiers to develop long-sought therapeutic approaches.

[0051] The examples also show that the toolkit of chemogenetically-activated depalmitoylases could be leveraged to develop depalmitoylation feedback circuits that alter protein localization in response to specific cell signaling events. FacePalm deployed in cultured neurons demonstrated bidirectional modulation of neuronal function by depalmitoylation. Thus, FacePalm is a powerful and versatile approach for targeted depalmitoylation of proteins and opens new avenues to study dynamic regulation of protein function by this post-translational modification.

[0052] In another aspects, a modified depalmitoylase that allows depalmitoylation of proteins in response to Erk phosphorylation is disclosed (dePalm-Er) is disclosed. Ras (KRas, NRas, HRas) is the most frequently mutated gene family in cancers. Mutant but not wild-type NRas upregulate the Ras-Mek-Erk pathway leading to increased cell proliferation and differentiation. However, developing allele- specific Ras inhibitors has been challenging. As Ras is only active when localized to cell membrane, one approach has been to develop inhibitors of Ras lipid modifications such as famesyl transferase inhibitors. However, these agents have been clinically disappointing. More recently, covalent inhibitors of the KRas^G12C variant were developed with the ability to disrupt cell viability in mutant cell lines and xenograft models. Both NRas and HRas undergo dynamic palmitoylation/dcpalmitoylation suggesting that palmitoylation inhibitors may have potential utility. Nevertheless, to date, it has been difficult to selectively target palmitoylation of mutant NRas and HRas proteins. Thus, dePalm-Er is a strategy to disrupt the pathological overactivation of the Erk pathway.

[0053] dePalm-Er comprises a depalmitoylase from a/p hydrolase domain-containing protein family, a WW phospho-binding domain, and an Erk substrate with an FQFP ERK-docking site. The WW phospho-binding domain is linked to the N-tenninus of the depalmitoylase from a/p hydrolase domain-containing protein family (N-terminal component of dePalm-Er). The Erk substrate with an FQFP ERK-docking site is linked to the C-terminus of the depalmitoylase (C- terminal component of dePalm-Er). Erk activation phosphorylates the Erk substrate in the C- terminal component of dePalm-Er, which in turn binds to the WW phospho-binding domain and thereby reconstitute the depalmitoylase. In some aspects, dePalm-Er comprises an Erk substrate from cdc24C. In certain embodiments, the depalmitoylase from a/p hydrolase domain-containing protein family is from the Abhdl7 family. In particular embodiments, dePalm-Er comprises Abhdl7C, a WW phospho-binding domain, and an Erk substrate with an FQFP ERK-docking site. [0054] Indeed, experimentally it was found that dePalm- Er promoted Golgi localization of mutant but not wild-type NRas, thus furnishing an alternative strategy to develop allele- specific Ras inhibitors with potential clinical utility. Of note, in experiments, dePalm-Er, although activated by Erk could still target a wide-range of proteins. Accordingly, methods of inhibiting Ras activity and/or pathological overaction of the Erk pathway with dePalm-Er are described. In some aspects, the targeting abilities of dePalm-Er may be enhanced by introducing selective nanobodies targeting NRas.

[0055] In still another aspects, an optogenetic actuator to manipulate protein palmitoylation (opto- depalm) is disclosed. This modified depalmitoylase comprises “enhanced magnets” derived from a photoreceptor, wherein the enhanced magnets are a protein pair that rapidly dimerize in the presence of a light signal. One member of the protein pair is attached to the N-terminal region fragment of a depalmitoylase from a/p hydrolase domain-containing protein family, while the other member is attached to the catalytic domain fragment of the depalmitoylase. In some embodiments, the enhanced magnets arc derived from photoreceptor Vivid from Neurospora crassa and comprises protein pairs eMagA and eMagB which rapidly dimerize in the presence of blue-light and reverses in the absence of blue-light. eMagA is attached to the N-terminal region fragment, and cMagB is attached to the catalytic domain fragment. In certain embodiments, the depalmitoylase from a/p hydrolase domain-containing protein family is from the Abhdl7 family. In particular embodiments, opto-depalm comprises Abhdl7C, eMagA, and eMagB. The Abhdl7C enzyme is split into two fragments: (1) the Abhdl7C N-terminal region, which attached to eMagA that serves as a membrane targeting moiety and (2) Abhdl7 C-terminal region which contains the catalytic domain region and is attached to eMagB.

[0056] As shown in the examples, the presence of opto-depalm with eMagA and eMagB without blue light did not alter the target protein’s typical membrane localization, while providing blue light changes the target protein’s localization to the cytosol. Interesting, removal of the blue light enables the target protein’s primary localization to be returned to the membrane. Thus, opto- depalm can reversibly manipulate protein palmitoylation. In certain implementations, the target protein or target protein complex is an acyl-transferase. In some embodiments, the target protein is CaVp2A.

[0057] Methods of depalmitoylating a target protein in a cell are also described. In one aspect, the method comprises providing FacePalm to the cell and providing rapamycin or a derivative thereof to the cell. In another aspects, method comprises providing dePalm-Er and inducing Erk phosphorylation in the cell. Such methods may be used for depalmitoylation of NRas thus inhibiting NRas activity and pathological overactivation of the Erk pathway. In yet another aspect, the method comprises providing opto-depalm to the cell and providing a light signal to the cell. Where opto-depalm comprises eMagA and eMagB as the “enhanced magnet”, the light signal is blue light.

EXAMPLES

[0058] The present disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

Example 1. Design of a chemogenetically-activated depalmitoylase [0059] To rationally design a chemogenetically-activated depalmitoylase, the recently identified Abhdl7 family of acyl-protcin thiocstcrascs was leveraged. This class of enzymes contain two key domains: (1) a carboxy-terminal (CT) a/p hydrolase domain that forms the catalytic domain responsible for hydrolyzing palmitoyl groups from target proteins, and (2) an amino terminal (NT) region that is itself palmitoylated and serves to recruit the enzyme to relevant membrane domains (FIG. 1A). Functionally, either mutations in the catalytic domain or deletions of the NT abolish Abhd 17 -mediated depalmitoylation. Given this unique molecular architecture, it was hypothesized that the two functional domains may be split, rendering the enzyme basally inactive (FIG. IB). The holoenzyme may then be reconstituted using a chemically inducible dimerization system (FIG. IB), thereby activating protein depalmitoylation with temporal precision. The rapid and irreversible rapamycin-induced dimerization of FK506 binding protein (FKBP)and rapamycin binding domain (FRB)was utilized. More specifically, to engineer a chemogenetically-activated depalmitoylase, the Abhdl7c enzyme was split and attached FRB to the N-terminal region and FKBP to the C-terminal region containing the catalytic domain, as shown in FIG. IB. At baseline, the catalytic domain is inactive. Upon addition of rapamycin, FKBP and FRB dimerizes activating the Abhdl7c enzyme. This overall strategy was termed: FKBP/FRB-dimerization activated chemogenetic excision of palmitoylation by Abhd 17c or FacePalml7C. AlphaFold predicted structures of Abhdl7A-Abhdl7c showed high structural similarity throughout the enzyme, with some divergence immediately following the amino-terminus, suggesting that this region may be well suited for splitting the holo-enzyme (FIGS. 1C and 2A-2C). Accordingly, FRB was attached to an N-terminal fragment and cyan fluorophore yielding FacePalml7CNT and FKBP to the CT of Abhdl7c and mCherry yielding FacePalml7Ccr (FIGS. IB and 1C), and the two components were expressed bicistronically along with mCherry as an expression marker. Importantly, previous studies have shown that membrane localization of Abhdl7 catalytic subunit is insufficient to restore its ability to robustly depalmitoylate target proteins. Here, FacePalml7C instead relies on reconstituting the holoenzyme using the chemically induced dimerizer.

[0060] To test functionality of FacePalml7C, the CavP2A subunit was considered, which is dually palmitoylated in its amino terminus, which allows for its segregation to the inner leaflet of the plasma membrane. The CavPiA subunit was chosen as a target for its important physiological role in tuning Cav channel dynamics. CavP2A was tagged with a yellow fluorescent protein (YFP) to monitor its localization using confocal microscopy. When expressed alone, CavP2A is enriched at the plasma membrane consistent with high levels of basal palmitoylation (FIG. ID). Co-expression of CavP2A with wild-type Abhdl7c but not catalytically inactive Abdhl7c S/A mutant resulted in redistribution of CavP2A throughout the cytoplasm (FIGS. ID, 2E, and 2F), consistent with its depalmitoylation and separation from the plasma membrane. In like manner, co-expression of Abhdl7a and Abhdl7b also resulted in cytoplasmic localization of CavP2A (FIGS. 2E-2F). These findings suggest that palmitoylation of CavP2A is tuned by the Abhdl7 class of enzymes. Changes in CavP2A localization with the engineered depalmitoylase FacePalml7C were next considered (FIG. IE). At baseline, it was found that CavP2A largely preserved its membrane localization (FIG. IE), consistent with low basal activity of the split enzyme. Addition of rapamycin resulted in the rapid recruitment of the FacePalml7CcT (red) to the FacePalml7CNT (blue) fragment (FIG. IE). The time course of change in FacePalml7CcT localization was quantified by measuring Pearson's correlation between the FacePalml7CcT and the FacePalml7CNT fragments (FIG. IF). Subsequently, a gradual cytoplasmic accumulation of CavP2A was observed (FIG. IE, bottom row). The time course of change in CavPiA localization following activation of FacePalml7C by rapamycin was quantified by measuring changes in cytosolic fluorescence normalized to initial cytosolic fluorescence (FIG. 1G). The time constant for the increase in cytosolic fluorescence was ~50 mins. This change is slower than the kinetics of rapamycin-induced dimerization, which is saturated within 1 min and is in line with previously reported depalmitoylation dynamics of various proteins. To further validate this metric, co-localization was monitored with a fluorescent membrane marker (CellMask) (FIGS. 10A-10C) by quantifying Pearson’s correlation (r_mem). FIG. 10D shows that rapamycin addition resulted in a decrease in r_mem indicated by reduced membrane localization. In addition, the time course for decrease in rmem closely matched the increase in cytosolic fluorescence (AF/FO) with no significant differences in the estimated time constants (FIGS. 10E-10G). This change in cytosolic fluorescence of was notably absent in (1) cells lacking FacePalml7C expression (FIG. 10H), (2) when co-expressed with a catalytically inactive FacePalml7C S/A mutant (FIGS. 1G and 101), or (3) in the presence of Palmostatin B, a pharmacological antagonist of Abhdl7 (FIGS. 10J and 10K). These results suggest that the catalytic activity of FacePalml7C is required to elicit a dynamic change in Cav02A localization. Furthermore, to exclude the possibility that the changes in CavP2A localization observed with FacePalml7C is due to a failure to palmitoylate newly synthesized proteins, cycloheximide was used to block protein synthesis. Even still, activation of FacePalml7C elicited a robust increase in cytosolic fluorescence of CavP2A, suggesting that the engineered depalmitoylases can act on existing proteins (FIGS. 10L and 10M). To further corroborate that changes in localization corresponds to altered CavP2A palmitoylation, an Acyl-RAC assay was used to directly detect the palmitoylated fraction. In cells co -transfected with YFP-tagged CavP2A and FacePalm, incubation with rapamycin for 4 h yielded an -60% reduction in palmitoylation (FIGS. 1H and II). These findings demonstrate that FacePalm serves as a chemogenetic approach to manipulate protein palmitoylation in live cells.

[0061] Whether FacePalm can evoke dynamic changes in the Ca²⁺ channel function through depalmitoylation of the CavP2A subunit was investigated. The Cav2.2 channel is functionally important for initiating presynaptic vesicle release in both the central and peripheral nervous system. HEK293 cells were co-transfected with Cav2.2 and auxiliary subunits 0.261 and CavP2A subunits. Whole-cell voltage-clamp recordings showed rapid activation and minimal inactivation of Cav2.2 in the presence of CavP2A (FIG. 1J, top). Addition of rapamycin yielded no change in current dynamics (FIG. 1 J, top). When FacePalm was co-transfected, the baseline currents remained unperturbed (FIG. 1J, bottom). However, addition of rapamycin yielded a marked increase in inactivation (FIG. 1J, bottom), consistent with previous studies where palmitoylation was disabled through mutagenesis of the Cav02A subunit. Population rsoo data measuring the fraction of peak current remaining following 800 ms of depolarization further confirmed this trend (FIG. IK). In all, these findings suggest that FacePalm is a powerful approach to dynamically tune protein palmitoylation to delineate molecular and physiological consequences.

Example 2. FacePalm is a generalizable strategy for chemogenetic protein depalmitoylation

[0062] Given that both Abhdl7a and Abhdl7b share a similar architecture as Abhdl7c, it was explored whether the FacePalm approach may be generalizable. Accordingly, Abhdl7a and Abhdl7b were split at analogous positions to Abhdl7c and attached FRB and FKBP to the NT and CT regions, thus yielding a toolkit of chemogenetic depalmitoylases. Whether rapamycin activation of FacePalml7A altered palmitoylation of C vP2A was probed using an acyl-RAC assay (FIGS. 3A and 3B). As with FacePalml7C, a strong reduction in the palmitoylated fraction of CavP2A was observed (FIG. 3B). Subsequently, the effect of rapamycin-induced activation of FacePalml7A on CavP2A localization was examined. It was found that with rapamycin activation, FacePalml7A evoked a substantial increase in cytosolic fluorescence of CavP2A , albeit blunted compared to FacePalm17C (FIGS. 3C and 3D Similar analysis confirmed functionality of FaccPalml7B in dcpalmitoylating (FIGS. 3E and 3F) and altering localization of CavPiA (FIGS. 3G and 3H). These findings illustrate the generality of this approach for inducible activation of Abhdl7 enzymes.

[0063] Beyond CavPiA, a staggering array of proteins with widely varying physiological functions are known to be palmitoylated. Here, it was considered whether FacePalm might alter localization of three unrelated proteins whose palmitoylation is thought to be important for its function. The first is (1) Kchip2, a Ca²⁺-binding protein that regulates Kv4 ion channels. The second is Lyn, a member of the Src family of tyrosine kinases. The third is NRas, a small G-protein involved in cell signal transduction and a proto-oncogene. Given that FacePalml7C evoked the strongest change in CavP2A localization, its effect was probed on these palmitoylated proteins. For both Kchip2 and Lyn, acyl-RAC RAC assays revealed statistically significant reduction in palmitoylation (Kchip2, FIGS. 31 and 3 J; Lyn, FIGS. 3M and 3N), and confocal imaging showed increased cytosolic localization of both Kchip2 (FIGS. 3K and 3L) and Lyn (FIGS. 30 and 3P) following activation of FacePalml7C. In similar manner, it was found that activation of FacePalml7C resulted in in reduced palmitoylation of NRas (FIGS. 3Q and 3R) and increased internalization of NRas, likely reflecting its accumulation in the Golgi complex as expected following depalmitoylation. Changes in NRas dynamics were quantified here by measuring the total area of intracellular puncta (FIGS. 3S and 3T). Collectively, these results highlight the generality of FacePalm as a toolkit for chemogenetic manipulation of palmitoylation for a wide range of proteins.

Example 3. Organelle-specific depalmitoylation by engineering FacePalm

[0064] Subcellular localization has emerged as a key contributor for specificity and for determining the biological consequences of protein palmitoylation. More concretely, various palmitoylating enzymes (zDHHCl-23) are localized to distinct intracellular organelles such as the endoplasmic reticulum, the Golgi, and the plasma membrane, leading to distinct biological outcomes. Consequently, new strategies to preferentially manipulate protein palmitoylation in distinct intracellular compailments are highly sought after. As FacePalm is genetically encoded, this approach is highly amenable to targeting distinct subcellular localizations. As such, it was reasoned that the N-terminal localizing component of FacePalml7C (FacePalm 17CNT) may be redirected to various subcellular locations via well-established subcellular targeting (FIG. 4A). The addition of rapamycin would then recruit FacePalm 17CCT, i.e. the catalytic domain of Abhdl7c, to the intended organelle, allowing dcpalmitoylation to proceed preferentially from this compartment. A library of FacePalm 17CNT valiants was generated containing targeting sequences to the (1) endoplasmic reticulum (ER), (2) Golgi, (3) plasma membrane (PM), (4) early endosomes, and (5) late endosomes (FIG. 4A). It is important to note that with this strategy, the FacePalml7Ccr fragment, which contains the catalytic subunit, remains the same. These variants were co-expressed with FacePalml7Ccr and assessed changes in membrane of Cav02A.

[0065] Upon rapamycin activation, non-targeted FacePalml7Ccr is broadly recruited to the PM and endosomal compartments. The degree of colocalization with each subcellular domain was quantified by determining Pearson’s correlation with a marker for respective subcellular locus (FIGS. 12A, 12D, 12G, 12J, and 12M). These changes are largely consistent with the localization of Abhdl7 holoenzyme as established by previous studies. By contrast, with subcellularly targeted FacePalml7C, rapamycin activation results in strong recruitment of FacePalml7CcT to desired compartments (FIGS. 12B, 12E, 12H, 12K, and 12N), as confirmed by a statistically significant increase in Pearson’s correlation with independent markers for each organelle (FIGS. 12C, 12F, 121, 12 L, and 120). Functionally, with ER-localized FacePalm, essentially no change was found in normalized cytosolic fluorescence of Cav02A (FIGS. 4B, 4C, and 4L) despite robust recruitment of the FacePalml7Ccrto this domain. By comparison, Golgi-localized FacePalm evoked a modest increase in normalized cytosolic fluorescence of CavP2A (FIGS. 4D, 4E, and 4L). Interestingly, both PM-localized (FIGs. 4F and 4G) and early endosome-localized (FIGS. 4H and 41) FacePalm evoked a still larger increase in cytosolic fluorescence (FIG. 4L). However, localizing FacePalm to late endosomes resulted in a modest reduction in cytosolic fluorescence compared to early- endosome localized FacePalm (FIGS. 4I-4L). The varying degree of increase in cytosolic fluorescence of CavP2A upon targeting FacePalm to distinct compartments points to heterogeneity in the efficacy of dcpalmitoylation from various intracellular organelles. In all, these results highlight the exquisite spatial and temporal precision with which FacePalm can manipulate protein palmitoylation.

Example 4. Engineering EacePalm to target specific protein complexes

[0066] Depalmitoylating enzymes such as Abhdl7 are often promiscuous and act on a wide range of cellular targets. As such, broad activation of FacePalm could contribute to global changes in the palmitoylome, potentially resulting in off-target effects and complex physiological outcomes. As such, it was considered whether FaccPalm may be engineered to selectively target individual protein complexes. One method is to attach FacePalml7CNT to a selective nanobody that binds to a specific target. The addition of rapamycin will then recruit FacePalml7Ccr to the target complex, resulting in its depalmitoylation (FIG. 13 A). This strategy was named FacePalm-individually targeted or FacePalm-it. To test whether this design could enable target specificity, Kchip2-tagged to YFP and Cav02A-tagged to a CFP were considered. Both proteins are robustly depalmitoylated by FacePalml7C (FIGS. IE- II and 3K-3L). To selectively tune palmitoylation of Kchip2, a previously published YFP-targeting nanobody was attached to FacePalml7CNT and co-expressed it with FacePalml7Ccr. Rapamycin activation of FacePalm-it resulted in a robust time-dependent increase in normalized cytosolic fluorescence of Kchip2, but not CavP2A (FIGS. 5B-5D), consistent with selective depalmitoylation of Kchip2.

[0067] Next, it was considered whether CavP2A can be selectively depalmitoylated while sparing Kchip2. FacePalml7CNT was modified with a selective high affinity nanobody for CavPiA (nb.F3) and co-expressed it with FacePalml7Ccr (FIGS. 5E-5F). Here, Kchip2 was tagged with YFP, while CavP2A was tagged with an mCherry fluorescent protein for convenience. It was found that the addition of rapamycin resulted in a strong increase in normalized cytosolic fluorescence of CavP2A without a corresponding increase in the cytosolic fluorescence of Kchip2 (FIG. 5F), consistent with selective depalmitoylation of CavP2A. Taken together, these findings illustrate the unprecedented selectivity of FacePalm-it for protein depalmitoylation.

[0068] While FacePalm-it allows inducible depalmitoylation of targeted proteins, in some cases, it may be advantageous to constitutively promote depalmitoylation of proteins. It was reasoned that directly attaching the catalytic subunit of Abhdl7c to a target selective nanobody could enable the constitutive depalmitoylation of the desired target protein. To test this possibility, the YFP- binding nanobody was attached to Abhdl7c catalytic domain (FIG. 5B) yielding Nb(YFP)-17c_Cat (FIG. 6A). Co-expression of this fusion protein with YFP-tagged CavP2A, KChip2, Lyn, NRas, and Gaq resulted in decreased membrane localization of these proteins (FIGS. 6B-6F). Furthermore, to probe whether this approach confers target selectivity YFP-tagged Kchip2 and mcherry-tagged CavP2A were co-expressed along with either (i) YFP-targeting nanobody tethered to Abhdl7Ccr (Nb(YFP)-17Cd) or (ii) CavP2A-targeting nanobody tethered to Abhdl7Ccr (Nb(CavP2A)-17Ccr). At baseline, both Kchip2 and CavP2A exhibit strong membrane localization (FIG. 5G). However, with Nb(YFP)-17CcT, Kchip2 is largely cytosolic while CavP2A is membrane localized (Fig. 5H). By contrast, with Nb(CavP2A)-17Ccr, Kchip2 remains membrane localized while CavP2A is cytosolic (Fig. 51). These changes were quantified by measuring the ratio of cytosolic to total fluorescence of either CavP2A or Kchip2 (F_cyt(CavP A) and F_cyt(Kchip2)) and calculating the ratio C, - F_cyt(C vP2A) I F_cyt(Kchip2) (Fig. 5J).This overall approach may be generalized to either constitutively or inducibly depalmitoylate any target by developing selective nanobodies or other synthetic binding proteins.

Example 5. Bidirectional modulation of neuronal activity by FacePalm

[0069] As multiple depalmitoylases are often co-expressed in a given cell type, it is unclear whether these enzymes are functionally redundant or may hydrolyze distinct substrates to evoke distinct physiological outcomes. For members of the Abhdl7 family, the structural similarity suggests that these proteins may have similar target profile, indicating that they may serve redundant functions. Distinct Abhdl7 proteins can bidirectionally tune excitability of cultured hippocampal neurons.

[0070] Protein palmitoylation is thought to be critical for neuronal function and regulation. Indeed, at the molecular level, a wide range of neuronal ion channels and synaptic proteins undergo palmitoylation that tunes their localization and function. Interestingly, all three Abhdl7 enzymes are expressed in neurons, albeit to varying degrees. This overlapping expression profile raises two distinct possibilities: (1) various Abhdl7 enzymes may subserve redundant functions or (2) distinct Abhdl7 enzymes may differentially target various proteins thereby evoking distinct changes in neuronal function. To distinguish between these possibilities, all three FacePalm variants were deployed individually in cultured rat hippocampal neurons using viral transduction. To first establish that all three FacePalm variants are functional in neurons, their effect was probed on the clustering and localization of PSD-95, a well-established target of the Abhdl7 class of enzymes, using immunohistochemistry and confocal microscopy. Without FacePalm, the addition of rapamycin yielded no appreciable change in spine density (FIGS. 8 A and 8B). However, rapamycin-induced activation of all three FacePalm variants resulted in a strong reduction in PSD95 puncta density along neuronal process (FIGS. 9A-9F). To ensure that changes in PSD95 puncta density indeed stem from enzymatic depalmitoylation, the enzymatic activity of FacePalml7A through FacePalml7C was disabled by introducing the S/A mutation in the catalytic triad. Reassuringly, the addition of rapamycin resulted in no apparent changes in PSD95 clustering in all three cases (FIGS. 13A-13F) These results confirm the functionality of FaccPalm in neurons and highlight its potential utility to manipulate protein palmitoylation in a native setting.

[0071] Thus assured, the effect of FacePalm variants on neuronal excitability using current clamp recordings was probed. The action potential (AP) firing rate evoked in response to a Is pulse to a family of current amplitudes (Zinj) was measured. In cells expressing FacePalml7A, no change in AP firing rate with rapamycin activation was found (FIGS. 9G and 9H), similar to cells lacking FacePalm (FIGS. 8C and 8D). When FacePalml7B is overexpressed, however, a marked increase in AP firing rate was found (FIGS. 91 and 9J), suggesting that FacePalml7B can upregulate neuron AP firing. In sharp contrast, when FacePalml7C is activated, an overall decrease in AP firing rate was observed (FIGS. 9K and 9L). To ensure that these changes in neuronal AP firing properties are due to altered palmitoylation, both catalytically-inactive FacePalml7B S/A mutant and FacePalml7C S/A mutant were expressed, no appreciable change in neuronal AP firing rates in both cases (FIGS. 13G-13I). As neuronal action potentials are coordinated by multiple ion channel complexes, changes in neuronal firing properties here could reflect altered regulation of one or more ion channel complexes. Indeed, several ion channel pore-forming subunits as well as auxiliary proteins have been shown to be palmitoylated.

[0072] While FacePalml7B increases action potential firing rate, FacePalml7C decreased neuronal activity. This suggests that although these enzymes are homologous and have overlapping targets, they may also act on disparate proteins leading to distinct physiological outcomes. Consistent with this possibility, recent studies have shown that STREX BK channels are depalmitoylated by both Abhdl7A and Abhdl7C but are largely insensitive to Abhdl7B. Beyond this, voltage-gated sodium channels and calcium channels are palmitoylated; however, whether Abhdl7 family targets these proteins is unknown. In the case of altered AP dynamics, it is likely that multiple channel subunits are differentially regulated by Abhdl7 resulting in overall changes in excitability.

[0073] Given its potentially broad impact a wide range of proteins, targeted depalmitoylation to tune specific aspects of neuronal function would be advantageous. Accordingly, it was sought to determine whether inducibly localizing a depalmitoylase to PSD95 may reduce its clustering without impacting AP firing properties which presumably reflect the effect of various ion channels. To this end, PSD95 binding fibronectin intrabodies generated by mRNA display (FingR) have been developed for imaging and manipulating PSD95 and postsynaptic function in neurons. Following the strategy with FaccPalm-it, PSD95-FingR was attached to FaccPalmN and bicistronically expressed FacePalmC. In addition, mcherry was used as an expression marker to identify cells expressing the two FacePalm components. At baseline, robust clustering of PSD95 quantified as the puncta density along neuronal processes was observed (FIG. 9M). In the presence of rapamycin, PSD95 clustering is significantly reduced, confirming the functionality of the targeted depalmitoylases (FIG. 9N). To probe changes in neuronal firing properties, current clamp recordings of hippocampal neurons expressing the targeted depalmitoylases were performed. Reassuringly, minimal changes in AP firing properties following rapamycin activation of the targeted depalmitoylases were found (FIGS. 90 and 9P).

[0074] The cultured hippocampal neurons experiments revealed an unexpected bidirectional tuning of cellular excitability by distinct classes of Abhdl7 enzymes.

[0075] These findings highlight the exquisite utility of FacePalm to monitor the depalmitoylation dynamics of endogenously palmitoylated proteins and to delineate its physiological functions. Future studies that use sequential pharmacological dissection of various ion channel currents, known as “onion-peeling” and complementary mass spectrometric analysis could provide a systematic understanding of ion channel regulation by depalmitoylation.

Example 6. Cell-signaling programmable activation of depalmitoylation

[0076] From a pathophysiological perspective, manipulation of protein palmitoylation has emerged as an attractive therapeutic possibility. To this end, the ability to conditionally activate protein depalmitoylation in response to a cell signaling event that may be upregulated in pathological settings is highly desirable yet challenging. A prominent example involves activating mutations in NRas that are commonly observed in highly aggressive cancers associated with high mortality. Constitutive activation of NRas results in upregulation of MAPK and PI3K pathways that cause sustained proliferation and tumor progression. In this regard, palmitoylation of NRas is important for its localization and downstream signaling, and inhibitors of the NRas palmitoylation cycle have been proposed as potential therapies (FIG. 7A). Accordingly, it was sought to engineer a synthetic feedback loop that activates a depalmitoylase in response to Erk, a downstream effector of NRas, and, in so doing, inhibit NRas in a context dependent manner (FIG. 7A). To do so, the design principle of FRET based ERK biosensors was leveraged. The N-terminus of Abhdl7C was engineered with a WW phospho-binding domain and the C-terminus of Abhdl7C with an Erk substrate from cdc24C along with an FQFP ERK-docking site (FIG. 7B). This bipartite system was named: dePalmitoylation, Erk-activated (dePalm-Er). With low Erk activity, the two components will remain unbound. However, Erk activation will result in phosphorylation of the Erk substrate in the C-terminal component of dePalm-Er, which in turn will bind the WW phospho- binding domain and thereby reconstitute the holo-enzyme. To probe the functionality of this engineered depalmitoylase, confocal imaging was used to monitor the subcellular localization of both wild-type and Q61K mutant NRas that is highly prevalent in melanomas and adenocarcinomas. As depalmitoylated NRas accumulates in the Golgi, colocalization of YFP- tagged NRas with a red Golgi marker (mCherry-golgin) was monitored. At baseline, both wildtype (FIG. 7C, top) and Q61K mutant NRas (FIG. 7C, bottom) are largely localized to the plasma membrane with minimal overlap with the Golgi compartment. (FIG. 7C, left-top and left-bottom). Further quantification revealed low Pearson’s correlation between red and yellow fluorescence at baseline (Fig. 7D). Over-expression of Abhdl7C results in internalization of both wild-type and mutant NRas (FIG. 7C, middle top), as evident from increased co-localization with the Golgi marker (FIG. 7D). Co-expression of dePalm-Er with wild- type NRas largely preserved the robust membrane localization of NRas as evident from both confocal images (FIG. 7C, top right), and minimal change in the Pearson’s correlation (FIG. 7D). In sharp contrast, co-expression of Q61K mutant NRas with dePalm-Er resulted in increased Golgi localization (FIG. 7C, bottom right). Population data further confirmed this trend (FIG. 7D). These results suggest that dePalm-Er allows selective activation of the Abhdl7C depalmitoylase in response to elevated or constitutive Erk activity. This approach further provides a potential therapeutic strategy for selective inhibition of cancer-causing NRas mutants. More broadly, these findings illustrate the versatility of genetically encoded approach developed here for programmable activation of protein depalmitoylation in response to specific physiological stimuli.

Example 7. Oplogenetic and reversible manipulation of protein palmitoylalion

[0077] A key limitation of the FacePalm approach for manipulating protein palmitoylation is poor reversibility, owing to the ultra-high affinity of rapamycin for FKBP and the slow clearance of rapamycin from cells. Accordingly, it was sought to devise an optogenetic approach, whereby the dimerization of the N- and C-termini of the Abhdl7c depalmitoylase may be induced by light illumination and reversed in darkness. To do so, the recently engineered ‘enhanced Magnets,’ derived from the Vivid photoreceptor from Neurospora crassa was utilized. The enhanced Magnets are composed of a protein pairs eMagA and eMagB which rapidly dimerize in the presence of blue-light and reverses in the absence of blue-light. eMagA was attached to the Abhdl7 NT fragment, and eMagB was attached to Abhdl7 CT catalytic subunit. The resulting pair is referred to herein as: opto-depalm (optogenetically activated depalmitoylases) (FIG. 11 A). Changes in localization of mCherry-tagged CavP2A in the presence and absence of opto-depalm were monitored. Without opto-depalm, no changes in Cav 2A localization with blue light illumination were observed (FIGS. 11B and 11C). With opto-depalm, Cav02A is initially membrane localized. However, following blue light (488 nm) illumination, an increase in cytosolic localization of CavPiA was observed. Without blue-light, CavP2A appeared to accumulate near the plasma membrane (FIGS. 11B and 11C), consistent with reduced activity of the optogenetic depalmitoylase. These findings illustrate the suitability of optogenetically-activated depalmitoylases for reversible manipulate protein palmitoylation.

Example 8. Targeted depalmitoylation of presynaptic Ca²⁺ channels in pain

[0078] Cav channels are physiologically essential as they convey Ca²⁺ influx that is responsible for initiating synaptic vesicle release in neurons and sensory cells. The predominant Cav channels involved in transmitter release at central and peripheral nervous system are Cav2.1 and Cav2.2 (FIG. 14A). Both channels are multisubunit complexes composed of a pore-forming subunit, the auxiliary P subunit, and the 0128 subunit (FIG. 14B). To support their physiological functions, these channels are spatially localized to the active zone release sites through exquisite trafficking mechanisms, and, furthermore, their function is regulated by a bevy of molecular factors and signaling mechanisms, as elaborated below. Importantly, altered channel function and their positioning within the active zone can profoundly alter the timing and strength of synaptic output. Not surprisingly, dysfunction of these channels is linked to a wide range of severe human diseases. [0079] As the primary calcium channel responsible for initiating vesicle release in nociceptive neurons of the peripheral nervous system, pharmacological block of Cav2.2 has garnered considerable attention as a non-opioid approach for targeting chronic or neuropathic pain (Fig. 14A). Furthermore, in chronic pain conditions, the Cav2.2 channels have been shown to be upregulated. Indeed, FDA approved blockers gabapentin, pregabalin, and ziconotide all target Cav2.2 in the DRG neurons; however, these agents have important limitations including severe side effects. Alternate strategics to inhibit Cav2.2 arc therefore highly desired.

[0080] As shown in FIGS. 14A and 14B, Cav2.2 channels are palmitoylated in HEK cells and in neurons. Acyl-RAC shows basal palmitoylation of CaV2.2 isolated from mouse cortex (FIG. 14A). Recombinantly expressed Cav2.2-YFP in HEK293 cells also show robust palmitoylation (FIG. 14B). Co-expression of Abhdl7c, a depalmitoylase, reduces palmitoylation of Cav2.2 demonstrating that Abhdl7 is the enzyme responsible for Cav2.2 regulation (FIG. 14B).

[0081] As shown in the changes of Cav2.2 channel activity of FIG. 15, palmitoylation upregulates Cav2.2 function. Palmitoylation upregulates Cav2.2 currents by increasing channel activity (FIGS. 16A-16C). Depalmitoylation of Cav2.2 by Abhdl7c inhibits single channel activity (compare FIG 16A and FIG. 16B). Co-expression of catalytically inactive Abhdl7c S/A mutant does not alter CaV2.2 activity (compare FIG. 16A and 16C). Depalmitoylation of endogenous Cav2.2 results in reduced Ca²⁺ currents (FIGS. 17A-17C).

[0082] Targeted depalmitoylation of Cav2.2, using the FacePalml7C further comprising a nanobody, diminishes both endogenous Cav2.2 currents (FIG. 18B) and Cav2.2 whole cell current density compared to nb.F3-Abhdl7C_Cat S/A mutant (FIG. 18C). Thus, that targeted depalmitoylation inhibits Cav2.2 currents. Thus, the disclosed modified depalmitoylase, for example FacePalm, may be a therapeutic for treating chronic or neuropathic pain.

Example 9. Experimental Procedures a. Molecular Biology and virus construction

[0083] Constructs were designed to contain a fluorophore in the backbone. The Cavf a subunit (M8O5O5.1) was tagged with YFP or mCherry at the C-Terminus with GSG linker in the N3 vector. NRas (NCBI accession #NM_002524.5) and Q6 IK mutant of NRas were synthesized from Twist Biosciences with cutting sites Hindlll and Xbal and inserted into vector containing a N-Terminus- Venus. Kchip2 (XM_048505192.1) were synthesized from Twist Biosciences as DNA fragments with cutting sites Nhel and Sall and C-terminal YFP. Lyn was a gift from Jesse Boehm & William Hahn & David Root (Addgene plasmid # 82215). G protein alpha-q-GFP was a gift from Catherine Berlot (Addgene plasmid # 66080). These sequences were then inserted into plasmids containing a C-terminal YFP. The calcium channel [ a subunit (M8O5O5.1) was tagged with YFP at the N- Terminus. N-terminus of Abhdl7 (Abhdl7a: NM 145421 ; Abhdl7b: NM146096, Abhdl7c: NM 133722) (1-55 amino acid sequence) tagged with an FRB was cloned into a plasmid using cutting sites EcoRI and BamHI upstream of a Cerulean fluorophore. FKBP with C-Terminus of Abhdl7 (residues 56-end containing the catalytic site) was cloned downstream of an mCherry fluorophore using Hindlll and BamHI cutting sites. Similarly, N-terminus of Abhdl7c tagged with an FRB and GFP targeted nanobody was inserted into a pcDNA3.1 vector with Hindlll and EcoRI. All viruses were synthesized and obtained from Vector builder. Abhdl7 NT- tagged FRB and FKBP- tagged Abhdl7 CT, separated by a P2A sequence for bicistronic expression, were designed and packaged into lentiviral (FacePalml7a, b) or adenoviral (FacePalml7C) vectors containing an mCherry regulated by an EFl promoter. b. Cell culture and transfections

[0084] Cultures were maintained at 37°C, 5% CO2, and 95% humidity. HEK293 cells were maintained in Dulbecco’s Modified Eagle Medium containing 10% FBS, L-Glutamine (2mM), 1% Penicillin- Streptomycin and Gentamicin (50pg/ml). For electrophysiology, cells were plated on coverslips and transfected using calcium phosphate method; Cav2.2 (0.5-1 pg), C_av|32a(0.5- Ipg) and oc28 (0.5-lpg) and SV40 driven T-antigen (0.5-lpg) depending on expression and coexpressed with FACEPALM (0.4-0.7pg) depending on expression. For AcylRAC/Western Blot assay, cells were plated on 60mm dishes and T-antigen (0.5-lpg), C_avP2a (2.5pg) an Facepalm (0.5-lpg) were transfected using calcium phosphate method. For confocal microscopy, cells were plated in glass -bottomed chambered coverslips. Cells were transfected using polyethyleneimine method of transfection as previously reported⁷⁵; T-antigen (0.2-0.3pg), Cavf a (0.5-0.7pg), Kchip2 (0.5-0.7pg), N-Ras (0.1-0.5pg), Q61K-NRas (0.1-0.5pg) and Lyn (0.5-lpg), co-transfected with Facepalm (0.4-0.7pg). c. Acyl RAC assay/ Western Blotting

[0085] Acyl Resin Assisted Capture assay kit was purchased from Badrilla (K010-311). The assay was performed per the manufacturer’s instructions. In brief, cells were collected 48 hrs posttransfection, and free cysteines were blocked for 4 hrs using a thiol blocking agent in Buffer A, following which the proteins were precipitated using acetone. The proteins were resuspended in binding buffer and quantified using BCA assay. Same quantity of protein was loaded onto the beads for the comparison conditions. The resuspended protein was treated with the cleavage reagent in the presence of thiopropyl Sepharose beads. The palmitoylated fraction was eluted with the 2X SDS buffer provided, loaded onto 4-12% Bis-Tris gradient gels (NP0321BOX; Thermofisher) and western blotting was performed. The blots were probed with rabbit anti GFP (1:2000) (A01388-40; Genscript) overnight. The blots were treated with secondary anti-rabbit HRP and developed using chemiluminescence. Data was analyzed using Prism GraphPad 10; unpaired t-test was used to obtain statistics between no rapamycin and rapamycin groups. d. Neuronal culture

[0086] E18 Primary rat hippocampal neuronal culture kit was purchased from Brain bits (Transnetyx, SKU KTSDEHP) and cells were cultured as per manufacturer’s instructions. Briefly, the hippocampi were digested in papain (2mg/ml) in Hibernate (-Ca) for 10 minutes at 30°C. Following this the digested tissue was triturated and centrifuged at l lOOrpm for Imin at room temperature. The cells were resuspended in Neurobasal A media with B27 and Glutamax with glutamate(25|iM). Cells were plated at 16000 cells/cm² onto cell culture plates coated with poly- D-lysine. Cells were transferred to maintenance media (Neurobasal A media with B27 and Glutamax without glutamate) 4 days after plating. Media was then changed every 3 days. Cells were transduced on day 7 or day 10 and experiments were performed on day 13-16. e. Immunocytochemistry

[0087] Neurons were fixed in 4% PFA containing 4% sucrose and 20mM EGTA. Neurons were then permeabilized with 0.1% Triton X-100 and blocked with 10% FBS. Neurons were incubated with anti-rabbit PSD95 (1:500; Cell signaling) and anti-mouse mCherry (1:500) overnight and treated with secondary anti-rabbit Alexa Fluor 488 (Invitrogen) and anti-mouse Alexa Fluor 568 (Invitrogen) before mounting the coverslips onto glass slides with Anti-FADE mounting medium with DAPI. f Confocal microscopy i. Time-lapse Live microscopy [0088] 24 - 48 hrs post transfection, HEK293 cells in chamber slides were rinsed with PBS (with Ca²⁺ and Mg²⁺) and Tyrodc solution (125mM NaCl, 2.5mM KC1, 3mM CaCl₂, ImM MgCl₂, lOmM HEPES and 30mM Glucose; pH7.4) was added. The chamber slides were placed on a stage top incubator and maintained at 37 °C and 5% CO₂. Cells were imaged using a 40X lens objective mounted on a Nikon Ti Eclipse inverted microscope equipped with a Yokogawa CSU-X1 confocal spinning disk and an Andor Zyla sCMOS camera. YFP, mCherry and Cerulean were excited using 488, 568 and 407 lasers and the gain and laser intensity were maintained across multiple fields, throughout the time-lapse measurements. Two to three fields of view were chosen for each sample and imaged before adding rapamycin (-lOmin). Post-rapamycin, cells were imaged every 10 min up to 90-120 min. ii. Fixed cell imaging

[0089] Slides containing mounted coverslips were imaged using 100X/60X lens objective, and PSD95 , mCherry and DAPI were imaged using the 488, 568 and 407 lasers. Images were acquired as a Z-stack and maximum-intensity projections were generated. Images were analyzed using Fiji (Image J, Dendritic spine counter plugin). For live cell imaging, images were normalized to the background fluorescence and the fluorescence intensity was measured by drawing a region of interest across all the time points. NRas undergoes a palmitoylation/depalmitoylation that shuttles between plasma membrane and golgi. To analyze NRas depalmitoylation, the time scale images were converted to 8-bit and the threshold was adjusted to remove the cell outline and generate a mask. Analyze particles was used to calculate the area of the particles for each time scale measurement. For dePalm-Er analyses, Pearson’s correlation coefficient was calculated for Golgin (mCherry) and NRas/ Q61K (YFP) using the coloc 2 plugin. For neurons, PSD95 staining was quantified by using the dendritic spine counter plugin. g. Whole-cell electrophysiology

[0090] Whole-cell voltage-clamp recordings for HEK293 were collected at room temperature using an Axopatch 200B amplifier (Axon Instruments). Borosilicate glass pipettes (2-4Mohm) were pulled with a horizontal puller (P97; Sutter Instruments Co.) and fire-polished (Microforge, Narishige, Tokyo, Japan). Recordings were low-pass filtered at 2kHz and 70% series resistance and capacitance compensation. For CaV2.2 experiments, Internal solutions contained 135 mM CsMcSO3, 5mM CsC12, ImM MgC12, 4mM MgATP, 10 mM HEPES, 10 mM BAPTA, adjusted to 290-295 mOsm with CsMeSO3 and pH 7.4 with CsOH. The external solutions contained 140 mM TEA-MeSO3, 10 mM HEPES (pH 7.4) and 5 mM BaC12, were adjusted to 300 mOsm with TEA-MeSO3 and pH 7.4 with TEA-OH. Cells were held at a potential of -80mV and family test pulses from -80 to +50 mV, with repeat intervals of 20s was used. For neurons, current clamp experiments were performed using the same experimental setup. Internal solution contained K gluconate (130mM), EGTA (O.lmM), MgC12 (ImM), MgATP (2mM), HEPES (lOmM), NaCl (5mM), KC1 (l lmM), Na2Phosphocreatine (5mM); pH7.4, adjusted to 290-295 mOsm. And the external solution contained aCSf (NaCl 124mM, KC1 2.5mM, NaH2PO4 E2mM, NaHCO3 24mM, HEPES 5mM, glucose lOmM, CaC12 2mM, MgC12 ImM) is adjusted to 300-305m0sm, pH 7.4. Measurements were made using family current injections from -0.1 to 4.5 pA current. Custom MATLAB (Mathworks) software was used to analysis AP morphology and firing rate.

[0091] Many additional implementations are possible. Further implementations are within the CLAIMS.

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Claims

( What is claimed:

1. A modified depalmitoylase comprising: a depalmitoylase from a/p hydrolase domain-containing protein family, wherein the depalmitoylase comprises an N-terminal region fragment and a catalytic domain fragment; a FK506 binding protein (FKBP); and a FKBP rapamycin binding domain (FRB), wherein the FRB is linked to the N-terminal region fragment and the FKBP is attached to the catalytic domain fragment.

2. The modified depalmitoylase of claim 1 further comprises a subcellular targeting sequence, wherein the subcellular targeting sequence is attached to the N-terminal region fragment and directs the N-terminal region fragment to an intracellular target.

3. The modified depalmitoylase of claim 1 or 2, further comprising a nanobody, wherein the nanobody is attached to the N-terminal region fragment and the nanobody specifically targets the N-terminal region fragment to bind to a target protein or target protein complex.

4. The modified depalmitoylase of claim 1 or 2, further comprising a nanobody, wherein the nanobody is attached to the catalytic domain fragment and the nanobody is specifically targets the catalytic domain fragment to bind to a target protein or target protein complex.

5. A modified depalmitoylase comprising: a depalmitoylase from a/p hydrolase domain-containing protein family comprising an N-terminus and a C-terminus; a WW phospho-binding domain; and an Erk substrate with an FQFP ERK-docking site, wherein the WW phospho-binding domain is linked to the N-terminus of the depalmitoylase and the Erk substrate with an FQFP ERK-docking site is linked to the C-terminus of the depalmitoylase.

6. The modified depalmitoylase of claim 5, wherein the Erk substrate is from cdc24C.

7. A modified depalmitoylase comprising: a depalmitoylase from a/p hydrolase domain-containing protein family, wherein the depalmitoylase comprises an N-terminal region fragment and a catalytic domain fragment; and a protein pair that rapidly dimerize in the presence of a light signal, wherein one member of the protein of the depalmitoylase, while the other member is attached to the catalytic domain fragment of the depalmitoylase.

8. The modified depalmitoylase of claim 7, wherein the protein pair that rapidly dimerize in the presence of a light signal are eMagA and eMagB.

9. The modified depalmitoylase of claim 8, wherein eMagA is attached to the N-terminal region fragment of the depalmitoylase and eMagB is attached to the C catalytic domain fragment of the depalmitoylase.

10. The modified depalmitoylase of any one of claims 1-9, wherein the depalmitoylase from the Abhdl7 family.

11. The modified depalmitoylase of claim 10, wherein the depalmitoylase is Abhdl7C.

12. A method of producing a chemically activated depalmitoylase comprising: linking a FK506 binding protein (FKBP) to a catalytic domain fragment from a depalmitoylase from a/p hydrolase domain-containing protein family; and linking a FKBP rapamycin binding domain (FRB) to a N-terminal region fragment from the depalmitoylase from a/p hydrolase domain-containing protein family, wherein: the depalmitoylase from a/p hydrolase domain-containing protein family is modified to be chemically activated, and providing rapamycin or a derivative thereof to the chemically activated depalmitoylase induces heterodimerization of the catalytic domain fragment and the N-terminal region fragment to create a catalytically active heterodimer.

13. The method of claim 12, further comprising attaching a subcellular targeting sequence to the N-terminal region fragment.

14. The method of claim 12, further comprising attaching a selective nanobody to the N- terminal region fragment and/or the catalytic domain fragment.

15. A method of producing a modified depalmitoylase comprising: linking a WW phospho-binding domain to a depalmitoylase from a/p hydrolase domain-containing protein family at its N-terminus; and linking an Erk substrate with an FQFP ERK-docking site to the depalmitoylase from a/p hydrolase domain-containing protein family at its C-terminus.

16. A method of producing a modified depalmitoylase comprising: linking eMagA to a N-terminal region fragment from a depalmitoylase from a/p hydrolase domain-containing protein family; and linking eMagB to a catalytic don hydrolase domain-containing protein family, wherein: providing blue light to the modified depalmitoylase induces heterodimerization of the catalytic domain fragment and the N-terminal region fragment to create a catalytically active heterodimer.

17. A method of depalmitoylating a target protein in a cell, the method comprising: providing the modified depalmitoylase of any one of claims 1-4 to the cell; and providing rapamycin or a derivative thereof to the cell.

18. A method of depalmitoylating a target protein in a cell, the method comprising: providing the modified depalmitoylase of produced according to the method of any one of claims 12-14 to the cell; and providing rapamycin or a derivative thereof to the cell.

19. The method of claim 17 or 18, wherein the intracellular target or the target protein or target protein complex comprises NRas.

20. A method of inhibiting Ras activity in cells, the method comprising administering a modified depalmitoylase of claim 5 or 6 to the cells.

21. The method of claim 20, the cells exhibit pathological overactivity of Erk pathway.

22. The method of claim 21, wherein the administration of the modified depalmitoylase inhibits pathological overactivity of Erk pathway in the cells.

23. A method for manipulating neuronal synapses and excitability in a subject, the method comprising administering a modified depalmitoylase of any one of claim 1, 3, and 4 to the subject, wherein the modified depalmitoylase targets Cav2.2.

24. The method of claim 24, wherein administration of the modified depalmitoylase targets chronic or neuropathic pain.