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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.

Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein STIM1 physically interacts with plasma membrane protein Orai1 to induce calcium release-activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here we show that STIM1 is also rapidly and transiently S-acylated at cysteine 437 upon ER calcium store depletion. S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating CRAC channel assembly and function.

Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein stromal interaction molecule 1 (STIM1) physically interacts with plasma membrane protein Orai1 to induce calcium release-activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long-chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here using the acyl resin-assisted capture assay, we show that STIM1 is also rapidly S-acylated at cysteine 437 upon ER calcium store depletion. Using a combination of live cell imaging and electrophysiology approaches with a mutant STIM1 protein, which could not be S-acylated, we determined that the S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating the CRAC channel assembly and function.

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 (also known as ZDHHC21), a member of the DHHC family of mammalian protein acyltransferases, mediates T cell receptor-induced S-acylation of proximal T cell signaling proteins. Using Zdhhc21dep mice, which express a functionally deficient version of DHHC21, we show that DHHC21 is a Ca2+/calmodulin-dependent enzyme critical for activation of naïve CD4+ T cells in response to T cell receptor stimulation. We find that disruption of the Ca2+/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.

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.

The extracellular accumulation of amyloid β (Aβ) fragments of amyloid precursor protein (APP) in brain parenchyma is a pathological hallmark of Alzheimer's disease (AD). APP can be cleaved into Aβ on late endosomes/multivesicular bodies (MVBs). E3 ubiquitin ligases have been linked to Aβ production, but specific E3 ligases associated with APP ubiquitination that may affect targeting of APP to endosomes have not yet been described. Using cultured cortical neurons isolated from rat pups, we reconstituted APP movement into the internal vesicles (ILVs) of MVBs. Loss of endosomal sorting complexes required for transport (ESCRT) components inhibited APP movement into ILVs and increased endosomal Aβ42 generation, implying a requirement for APP ubiquitination. We identified an ESCRT-binding and APP-interacting endosomal E3 ubiquitin ligase, ubiquitination factor E4B (UBE4B) that regulates APP ubiquitination. Depleting UBE4B in neurons inhibited APP ubiquitination and internalization into MVBs, resulting in increased endosomal Aβ42 levels and increased neuronal secretion of Aβ42. When we examined AD brains, we found levels of the UBE4B-interacting ESCRT component, hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs), were significantly decreased in AD brains. These data suggest that ESCRT components critical for membrane protein sorting in the endocytic pathway are altered in AD. These results indicate that the molecular machinery underlying endosomal trafficking of APP, including the ubiquitin ligase UBE4B, regulates Aβ levels and may play an essential role in AD progression.

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.

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.

Oligomerization or homotypic clustering diverts Golgi membrane proteins into the canonical GGA1/clathrin-dependent Golgi-to-lysosome pathway revealing the presence of cellular quality control that could be useful for therapies designed to down-regulate specific proteins in the secretory pathway., Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130’s cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes.

During invasion of host cells the Shigella bacterial toxin sorts away from degradative lysosomes and traffics to the Golgi complex by binding the cycling transmembrane protein GPP130 as GPP130 returns from endosomes to the Golgi. Remarkably, an increase in Golgi manganese (Mn) alters the trafficking of GPP130 causing its degradation in lysosomes thereby protecting cells against lethal doses of purified toxin. Mn-induced down-regulation of GPP130 is thus an important paradigm for therapy against the incurable Shiga toxicosis as well as metal-regulated protein sorting in the Golgi. The experiments herein reveal significant aspects of the mechanism. We identified a transferable trafficking determinant within the GPP130 lumenal stem domain that bound Mn and observed that Mn binding induced its oligomerization in the Golgi. Significantly, alanine substitutions that blocked Mn binding abrogated both oligomerization and GPP130 sorting to lysosomes. GPP130 oligomers engaged the canonical Golgi-lysosomal trafficking machinery, as GPP130 exit from the Golgi required the sorting adaptor GGA1 and the clathrin vesicle coat complex. Surprisingly, oligomerization was sufficient since GPP130 redistributed to lysosomes in the absence of Mn by forced aggregation using a drug-controlled selfinteracting version of the FKBP domain. Further, neither the cytoplasmic domain nor residues in the lumenal stem domain critical for normal GPP130 cycling were required for this redistribution. These observations suggested that higher order oligomerization might generally cause lysosomal targeting of Golgi membrane proteins. Indeed, induced oligomerization of two other unrelated Golgi proteins caused their redistribution to endosome/lysosome-like punctae and subsequent degradation. The ER-based unfolded protein response was not activated during this process. Altogether, these results show that Mn binds and oligomerizes GPP130 targeting it to what may be a pre-existing quality control pathway in the Golgi where aggregated proteins are degraded in lysosomes.

This paper develops tools to examine selected major issues in the Indian economy. The study computes the potential growth rate of the economy and the agricultural sector, extends the analysis of the fiscal stimulus and its effects, and estimates the short and long run elasticities of India’s trade. This brings out the need for structural reforms in raising the potential growth rate of economy and that of agriculture to achieve a non-inflationary, high growth trajectory for the country. The fiscal stimulus effects indicate the importance of fiscal consolidation efforts to sustain high growth. The trade elasticities buttress the case for maintaining an appropriate real effective exchange rate.

Despite signs of recovery from the global financial crisis, the GDP growth rate for the Indian economy is likely to be between 5.8 to 6.1 per cent in 2009-10, below the 6.7 per cent recorded in fiscal 2008-09. While there has been an improvement in Indian industry, particularly the manufacturing sector, the adverse impact of the fall in kharif production due to a rainfall deficiency will act as a drag on the overall growth of the economy. In the current financial year, the major policy challenges for the government will come from the rather sharp rise in inflation and deteriorating public finances. The balance of payments situation may also require policy attention despite a narrowing of the current account deficit and a considerable capital account surplus because of the appreciation of the rupee.