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S-acylation – reversible post-translational lipidation of cysteine residues – is emerging as an important regulatory mechanism in T cell signaling. Dynamic S-acylation is critical for protein recruitment into the T cell receptor complex and initiation of the subsequent signaling cascade. However, the enzymatic control of protein S-acylation in T cells remains poorly understood. Here, we report a previously uncharacterized role of DHHC21, a member of the mammalian family of DHHC protein acyltransferases, in regulation of the T cell receptor pathway. We found that loss of DHHC21 prevented S-acylation of key T cell signaling proteins, resulting in disruption of the early signaling events and suppressed expression of T cell activation markers. Furthermore, downregulation of DHHC21 prevented activation and differentiation of naïve T cells into effector subtypes. Together, our study provides the first direct evidence that DHHC protein acyltransferases can play an essential role in regulation of T cell-mediated immunity.

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here, we investigated whether β-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that β-adrenergic stimulation led to rapidly increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and contractile responses. We identified the plasma membrane-localized palmitoyl acyltransferase DHHC5 as an important mediator of the stimulus-dependent palmitoylation in cardiomyocytes. Knockdown of DHHC5 showed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and functional responses downstream of β-adrenergic stimulation. A palmitoylation assay with purified components revealed that Gαs and Gαi are direct substrates of DHHC5. Finally, we provided evidence that the C-terminal tail of DHHC5 can be palmitoylated in response to stimulation and such modification is important for its dynamic localization and function in the plasma membrane. Our results reveal that DHHC5 is a central regulator of signaling downstream of β-adrenergic receptors in cardiomyocytes.

Zn2+has been shown to have a wide range of modulatory effects on neuronal AMPARs. However, the mechanism of modulation is largely unknown. Here we show that Zn2+inhibits GluA2(Q) homomeric receptors in an activity- and voltage-dependent manner, indicating a pore block mechanism. The rate of inhibition is slow, in the hundreds of milliseconds at millimolar Zn2+concentrations; hence, the inhibition is only observed in the residual nondesensitizing currents. Consequently, the inhibition is higher for GluA2 receptors in complex with auxiliary subunits γ2 and γ8 where the residual activation is larger. The extent of inhibition is also dependent on charge at site 607, the site that undergoes RNA editing in GluA2 subunits replacing glutamine to arginine, with the percent inhibition being lower and IC50being higher for the edited GluA2(R) relative to unedited GluA2(Q) and to GluA2(Q607E), a mutation observed in the genetic screen of a patient exhibiting developmental delays. We also show that Zn2+inhibition is significant during rapid repetitive activity with pulses of millimolar concentrations of glutamate in both receptors expressed in HEK cells as well as in native receptors in cortical neurons of C57BL/6J mice of either sex, indicating a physiological relevance of this inhibition.SIGNIFICANCE STATEMENTZn2+is present along with glutamate in synaptic vesicles and coreleased during synaptic transmission, modulating the postsynaptic ionotropic glutamate receptors. While Zn2+inhibition of the NMDA subtype of the ionotropic glutamate receptors is well characterized, the mechanism of modulation of the AMPA subtype is much less known. Here we have systematically studied Zn2+inhibition of AMPARs by varying calcium permeability, auxiliary subunits, and activation levels and show that Zn2+inhibits AMPARs in an activity-dependent manner, opening up this pathway as a means to pharmacologically modulate the receptors.

Despite the recognized significance of reversible protein lipidation (S-acylation) for T cell receptor signal transduction, the enzymatic control of this post-translational modification in T cells remains poorly understood. Here, we demonstrate that DHHC21, a member of the DHHC family of mammalian protein acyltransferases, mediates agonist-induced S-acylation of proximal T cell signaling proteins. Using Zdhhc21depmice expressing a functionally deficient version of DHHC21, we show that DHHC21 is a calcium/calmodulin-dependent enzyme critical for activation of naïve CD4+T cells in response to T cell receptor stimulation. We found that disruption of the calcium/calmodulin binding domain of DHHC21 does not affect thymic T cell development but prevents differentiation of peripheral CD4+T cells into Th1, Th2, and Th17 effector T helper lineages. Our findings identify DHHC21 as an essential component of the T cell receptor signaling machinery and define a new role for protein acyltransferases in regulation of T cell-mediated immunity.SignificanceThis study identifies DHHC21, a member of the DHHC family of mammalian protein acyltransferases, as a novel component of the TCR signaling pathway and demonstrates that this enzyme critically regulates activation and differentiation of CD4+T cells by mediating rapid TCR-induced S-acylation of signaling proteins. This finding shows that protein acyltransferases can play a vital function in regulation of T cell-mediated immunity and thus serve as potential drug targets in diseases associated with altered immune system homeostasis.

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Uterine leiomyomas or fibroids are the most common tumors of the female reproductive tract. Estrogen (E(2)), a steroid-derived hormone, and its receptors (ERs), particularly ER-α, are important drivers for the development and growth of leiomyomas. We previously demonstrated that simvastatin, a drug used for hyperlipidemia, also possesses anti-leiomyoma properties. The aim of this work is to investigate the impact of simvastatin on ER-α signaling in leiomyoma cells, including its expression, downstream signaling, transcriptional activity, post-translational modification, trafficking and degradation. Primary and immortalized human uterine leiomyoma (HuLM) cells were used for in vitro experiments. Immunodeficient mice xenografted with human leiomyoma tissue explants were used for in vivo studies. Leiomyoma samples were obtained from patients enrolled in an ongoing double-blinded, phase II, randomized controlled trial. Here, we found that simvastatin significantly reduced E(2)-induced proliferation and PCNA expression. In addition, simvastatin reduced total ER-α expression in leiomyoma cells and altered its subcellular localization by inhibiting its trafficking to the plasma membrane and nucleus. Simvastatin also inhibited E(2) downstream signaling, including ERK and AKT pathways, E(2)/ER transcriptional activity and E(2)-responsive genes. To explain simvastatin effects on ER-α level and trafficking, we examined its effects on ER-α post-translational processing. We noticed that simvastatin reduced ER-α palmitoylation; a required modification for its stability, trafficking to plasma membrane, and signaling. We also observed an increase in ubiquitin-mediated ER-α degradation. Importantly, we found that the effects of simvastatin on ER-α expression were recapitulated in the xenograft leiomyoma mouse model and human tissues. Thus, our data suggest that simvastatin modulates several E(2)/ER signaling targets with potential implications in leiomyoma therapy and beyond.

Chronic ER stress occurs when protein misfolding in the Endoplasmic reticulum (ER) lumen remains unresolved despite activation of the unfolded protein response. We have shown that traumatic injury such as a severe burn leads to chronic ER stress in vivo leading to systemic inflammation which can last for more than a year. The mechanisms linking chronic ER stress to systemic inflammatory responses are not clear. Here we show that induction of chronic ER stress leads to the release of known and novel damage-associated molecular patterns (DAMPs). The secreted DAMPs are aggregated and markedly protease resistant. ER stress-derived DAMPs activate dendritic cells (DCs) which are then capable of polarizing naive T cells. Our findings indicate that induction of chronic ER stress may lead to the release of hyperstable DAMPs into the circulation resulting in persistent systemic inflammation and adverse outcomes.

Chronic ER stress occurs when protein misfolding in the ER lumen remains unresolved despite activation of the unfolded protein response. We have shown that traumatic injury such as a severe burn leads to chronic ER stressin vivoleading to systemic inflammation which can last for more than a year. The mechanisms linking chronic ER stress to systemic inflammatory responses is not clear. Here we show that induction of chronic ER stress leads to the release of known and novel damage-associated molecular patterns (DAMPs). The secreted DAMPs are aggregated and markedly protease resistant. ER stress-derived DAMPs activate dendritic cells which are then capable of polarizing naïve T cells. Our findings indicate that induction of chronic ER stress may lead to the release of hyperstable DAMPs into the circulation resulting in persistent systemic inflammation and adverse outcomes.

ZAP-70 is a cytoplasmic tyrosine kinase essential for T cell-mediated immune responses. Upon engagement of the T cell receptor, ZAP-70 is quickly recruited to the specialized plasma membrane domains, becomes activated and released to phosphorylate its laterally segregated downstream targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in T cell receptor signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized reversible lipidation (S-acylation) of ZAP-70. We found that this post-translational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the T cell receptor signaling cascade suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with early T cell signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function.Significance StatementActivation of T cells is a critical part of the adaptive immune response to pathogen exposure. We found that ZAP-70, a regulatory protein essential for T cell activation, can undergo a post-translational modification with long chain fatty acids, known as S-acylation. In this report, we show that S-acylation of ZAP-70 is T cell receptor-dependent and required for its signaling function. We found that loss of ZAP-70 S-acylation resulted in T cell unresponsiveness to T cell receptor stimulation indicating that abnormalities in protein S-acylation can potentially contribute to the T cell immunodeficiency disorders.

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here we investigated whether β-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that β-adrenergic stimulation led to increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and positive inotropic responses. Additionally, we identified for the first time that G protein-coupled receptor kinase 2 (GRK2) is a palmitoylated protein in cardiomyocytes. The kinetics of GRK2 palmitoylation were distinct from those observed with Gαs and Gαi after β-adrenergic stimulation. Knockdown of the plasma membrane-localized palmitoyl acyltransferase DHHC5 revealed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and GRK2 and functional responses downstream of β-adrenergic stimulation. Our results reveal that DHHC5 activity is required for signaling downstream of β-adrenergic receptors.

Calcium ions regulate almost all cellular processes. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitous calcium channels which mediate calcium release primarily from endoplasmic reticulum stores. IP3Rs are activated by the soluble second messenger inositol 1,4,5-trisphosphate (IP3), which is produced in response to various stimuli by the action of phospholipase C enzymes. Very soon after the discovery that IP3 is a calcium mobilizing second messenger, it was found that T-cell receptor signaling is dependent upon IP3R activity. It is now known that cell signaling throughout the immune system is dependent upon IP3R-dependent calcium release for many critical processes, including cellular activation, proliferation, and death. This channel plays a critical role in orchestrating the immune response in health and disease and thus offers potential as a therapeutic target in immune-related disorders. WIREs Membr Transp Signal 2012,1:329–339. doi: 10.1002/wmts.27 For further resources related to this article, please visit the WIREs website.

An important role in the regulation of apoptotic calcium release is played by the ubiquitously expressed family of inositol 1,4,5-trisphosphate receptor (IP(3)R) channels. One model for IP(3)R activation during apoptosis is cleavage by the apoptotic protease caspase 3. Here we show that early elevations in cytosolic calcium during apoptosis do not require caspase 3 activity. We detected a robust increase in calcium levels in response to staurosporine treatment in primary human fibroblasts and HeLa cells in the presence of the caspase inhibitor Z-VAD, indicating that calcium release during the initiation of apoptosis occurs independently of caspase 3. Similar results were obtained with MCF-7 cells which lack caspase 3 expression. Stable expression of caspase 3 in MCF-7 cells and TAT-based transduction of the active recombinant caspase 3 directly into living MCF-7 cells had marginal effects on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. Significantly, blocking IP(3) binding to the IP(3)R with an IP(3) sponge resulted in suppression of staurosporine-induced calcium release and cell death. Collectively, our results suggest that generation of IP(3) is sufficient for the initiation of apoptotic calcium signaling, and caspase 3-mediated truncation of IP(3)R channel is a consequence, not causative, of apoptotic calcium release.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis(1). During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol(2). It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology(3). Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)(4) or aequorin(5) targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin(4). Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM(4). These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

Calcium is known to be an important second messenger in regulation of various physiological processes including execution of programmed cell death. Calcium release from endoplasmic reticulum (ER) triggers activation of multiple pro-apoptotic factors resulting in apoptosis progression and, ultimately, cell death. Apoptotic calcium release from ER stores is mediated by inositol 1,4,5-trisphosphate receptor (IP3R) channels. Different models have confirmed the importance of IP3R channels in modulation of apoptosis, however many details of IP3R-dependent calcium release still remain enigmatic. Previously, we have demonstrated that calcium release from IP3R channels in response to apoptotic stimuli occurs in two temporally distinct phases. A rapid oscillatory cytosolic calcium release during initiation of apoptotic signaling is followed by delayed and sustained increase in calcium levels associated with persistent opening of IP3R channels. Here we show that early elevations in cytosolic calcium do not require caspase-3 activity. Using MCF-7 cells deficient in caspase-3, we detected robust increase in calcium levels in response to staurosporine treatment indicating that calcium release during initiation of apoptosis occurs independently of caspase-3. To further determine contribution of caspase-3 in regulation of calcium release we generated a MCF-7 cell line stably expressing pro-caspase-3. Additionally, we used TAT-based transducing peptide to deliver active recombinant caspase-3 directly into living MCF-7 cells. Utilizing both methodologies, we found that caspase-3 has a marginal effect on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. These results suggest that caspase-3 mediated truncation of IP3R channels is a consequence, not causative, of apoptotic signaling events.