Partial loss of Sorting Nexin 27 resembles age- and Down syndrome-associated T cell dysfunctions.

BACKGROUND: Sorting Nexin 27 (SNX27)-retromer complex facilitates cargo recycling from endosomes to the plasma membrane. SNX27 downregulation in neurons, as the result of Trisomy 21 (T21), has been linked with cognitive deficits due to impairment of AMPA and NMDA receptor recycling. Studies in human T cell lines likewise demonstrated that SNX27 regulates the correct delivery of cargoes to the immune synapse limiting the activation of pro-inflammatory pathways. Nevertheless, the physiological consequences of partial SNX27 loss in T cell homeostasis are still unclear. RESULTS: In this study, we have explored the consequences of T cell specific partial SNX27 downregulation in mice. T cells with partial SNX27 deficiency show a marked deficit in the CD4+ T cell pool, a hallmark of aging in mice and humans, and a well-characterized comorbidity of individuals with Down syndrome (DS). When analyzed ex vivo, CD4+ T cells with partial SNX27 deletion demonstrate enhanced proliferation but diminished IL-2 production. In contrast, the CD8+ population show enhanced expression of pro-inflammatory cytokines and lytic enzymes. CONCLUSIONS: This mouse model supports the relevance of SNX27 in the organization of the immune synapse, previously described in cell lines, as well as in the control of T cell homeostasis. Individuals with DS experiment an acceleration of the aging process, which particularly affects the immune and central nervous systems. Thus, we hypothesize that reduced SNX27 expression in DS could contribute to the dysregulation of these systems and further research in SNX27 will shed light on the molecular factors underlying the phenotypes observed in people with DS and its contribution to aging.

Identification of Host PDZ-Based Interactions with the SARS-CoV-2 E Protein in Human Monocytes.

Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus's pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response.

Diacylglycerol kinase α inhibition cooperates with PD-1-targeted therapies to restore the T cell activation program.

BACKGROUND: Antibody-based therapies blocking the programmed cell death-1/ligand-1 (PD-1/PD-L1) axis have provided unprecedent clinical success in cancer treatment. Acquired resistance, however, frequently occurs, commonly associated with the upregulation of additional inhibitory molecules. Diacylglycerol kinase (DGK) α limits the extent of Ras activation in response to antigen recognition, and its upregulation facilitates hypofunctional, exhausted T cell states. Pharmacological DGKα targeting restores cytotoxic function of chimeric antigen receptor and CD8+ T cells isolated from solid tumors, suggesting a mechanism to reverse T cell exhausted phenotypes. Nevertheless, the contribution of DGKα downstream of the PD-1/PD-L1 inhibitory axis in human T cells and the consequences of combining DGKα and anti-PD-1/PD-L1 inhibitors are still unresolved relevant issues. MATERIALS AND METHODS: We used a human triple parameter reporter cell line to investigate DGKα contribution to the PD-1/PD-L1 inhibitory pathway. We also addressed the impact of deleting DGKα expression in the growth dynamics and systemic tumor-derived effects of a PD-1-related tumor model, the MC38 colon adenocarcinoma. RESULTS: We identify DGKα as a contributor to the PD-1/PD-L1 axis that strongly limits the Ras/ERK/AP-1 pathway. DGKα function reinforces exhausted T cell phenotypes ultimately promoting tumor growth and generalized immunosuppression. Pharmacological DGKα inhibition selectively enhances AP-1 transcription and, importantly, cooperates with antibodies blocking the PD-1/PD-L1 interrelation. CONCLUSIONS: Our results indicate that DGKα inhibition could provide an important mechanism to revert exhausted T lymphocyte phenotypes and thus favor proper anti-tumor T cell responses. The cooperative effect observed after PD-1/PD-L1 and DGKα blockade offers a promising strategy to improve the efficacy of immunotherapy in the treatment of cancer.

Essential function for the GTPase TC21 in homeostatic antigen receptor signaling.

T cell antigen receptors (TCRs) and B cell antigen receptors (BCRs) transmit low-grade signals necessary for the survival and maintenance of mature cell pools. We show here that TC21, a small GTPase encoded by Rras2, interacted constitutively with both kinds of receptors. Expression of a dominant negative TC21 mutant in T cells produced a rapid decrease in cell viability, and Rras2(-/-) mice were lymphopenic, possibly as a result of diminished homeostatic proliferation and impaired T cell and B cell survival. In contrast, TC21 was overexpressed in several human lymphoid malignancies. Finally, the p110delta catalytic subunit of phosphatidylinositol-3-OH kinase (PI(3)K) was recruited to the TCR and BCR in a TC21-dependent way. Consequently, we propose TC21 directly links antigen receptors to PI(3)K-mediated survival pathways.

Diacylglycerol kinase control of protein kinase C.

The diacylglycerol kinases (DGK) are lipid kinases that transform diacylglycerol (DAG) into phosphatidic acid (PA) in a reaction that terminates DAG-based signals. DGK provide negative regulation to conventional and novel protein kinase C (PKC) enzymes, limiting local DAG availability in a tissue- and subcellular-restricted manner. Defects in the expression/activity of certain DGK isoforms contribute substantially to cognitive impairment and mental disorders. Abnormal DGK overexpression in tumors facilitates invasion and resistance to chemotherapy preventing tumor immune destruction by tumor-infiltrating lymphocytes. Effective translation of these findings into therapeutic approaches demands a better knowledge of the physical and functional interactions between the DGK and PKC families. DGKζ is abundantly expressed in the nervous and immune system, where physically and functionally interacts with PKCα. The latest discoveries suggest that PDZ-mediated interaction facilitates spatial restriction of PKCα by DGKζ at the cell-cell contact sites in a mechanism where the two enzymes regulate each other. In T lymphocytes, DGKζ interaction with Sorting Nexin 27 (SNX27) guarantees the basal control of PKCα activation. SNX27 is a trafficking component required for normal brain function whose deficit has been linked to Alzheimer's disease (AD) pathogenesis. The enhanced PKCα activation as the result of SNX27 silencing in T lymphocytes aligns with the recent correlation found between gain-of-function PKCα mutations and AD and suggests that disruption of the mechanisms that provides a correct spatial organization of DGKζ and PKCα may lie at the basis of immune and neuronal synapse impairment.

Diacylglycerol Kinase Malfunction in Human Disease and the Search for Specific Inhibitors.

The diacylglycerol kinases (DGKs) are master regulator kinases that control the switch from diacylglycerol (DAG) to phosphatidic acid (PA), two lipids with important structural and signaling properties. Mammalian DGKs distribute into five subfamilies that regulate local availability of DAG and PA pools in a tissue- and subcellular-restricted manner. Pharmacological manipulation of DGK activity holds great promise, given the critical contribution of specific DGK subtypes to the control of membrane structure, signaling complexes, and cell-cell communication. The latest advances in the DGK field have unveiled the differential contribution of selected isoforms to human disease. Defects in the expression/activity of individual DGK isoforms contribute substantially to cognitive impairment, mental disorders, insulin resistance, and vascular pathologies. Abnormal DGK overexpression, on the other hand, confers the acquisition of malignant traits including invasion, chemotherapy resistance, and inhibition of immune attack on tumors. Translation of these findings into therapeutic approaches will require development of methods to pharmacologically modulate DGK functions. In particular, inhibitors that target the DGKα isoform hold particular promise in the fight against cancer, on their own or in combination with immune-targeting therapies.

Interplay Between SNX27 and DAG Metabolism in the Control of Trafficking and Signaling at the IS.

Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized contact between both cells named the immune synapse (IS). This highly organized structure ensures cell-cell communication and sustained T-cell activation. An essential lipid regulating T-cell activation is diacylglycerol (DAG), which accumulates at the cell-cell interface and mediates recruitment and activation of proteins involved in signaling and polarization. Formation of the IS requires rearrangement of the cytoskeleton, translocation of the microtubule-organizing center (MTOC) and vesicular compartments, and reorganization of signaling and adhesion molecules within the cell-cell junction. Among the multiple players involved in this polarized intracellular trafficking, we find sorting nexin 27 (SNX27). This protein translocates to the T cell-APC interface upon TCR activation, and it is suggested to facilitate the transport of cargoes toward this structure. Furthermore, its interaction with diacylglycerol kinase ζ (DGKζ), a negative regulator of DAG, sustains the precise modulation of this lipid and, thus, facilitates IS organization and signaling. Here, we review the role of SNX27, DAG metabolism, and their interplay in the control of T-cell activation and establishment of the IS.

Sorting nexin 27 interactome in T-lymphocytes identifies zona occludens-2 dynamic redistribution at the immune synapse.

T Lymphocyte recognition of antigens leads to the formation of a highly organized structure termed immune synapse (IS) by analogy with the neuronals synapse. Sorting nexin 27 (SNX27) controls the endosomal traffic of PSD95, Dlg1, ZO-1 (PDZ) domain-interacting proteins, and its alteration is associated with impaired synaptic function and neurological diseases. In T-lymphocytes, SNX27-positive vesicles polarize to the IS, the identity of SNX27 interactors in these conditions nonetheless remains unknown. Here we used proteomics to analyze the SNX27 interactome purified from IS-forming T cells, and confirmed the conserved nature of the SNX27/WASH/retromer association in hematopoietic cells. Furthermore, our comparative interactome analysis of SNX27 wild-type and a mutant-deficient for PDZ cargo recognition identified the epithelial cell-cell junction protein zona occludens-2 (ZO-2) as an IS component. Biochemistry and microscopy approaches in T cells confirmed SNX27/ZO-2 PDZ-dependent interaction, and demonstrated its role controlling the dynamic localization of ZO-2 at the IS. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in nervous and epithelial systems also participate in IS regulation.

Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals.

The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα-/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.

Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells.

Sorting nexin 27 (SNX27) controls the endosomal-to-cell-surface recycling of diverse transmembrane protein cargos. Crucial to this function is the recruitment of SNX27 to endosomes which is mediated by the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by its phox homology (PX) domain. In T-cells, SNX27 localizes to the immunological synapse in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide-lipid-binding capabilities of full-length SNX27, and discovered a new PtdInsP-binding site within the C-terminal 4.1, ezrin, radixin, moesin (FERM) domain. This binding site showed a clear preference for bi- and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the immunological synapse of activated T-cells, cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters the SNX27 distribution in both endosomal recycling compartments and PtdIns(3,4,5)P3-enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during immunological synapse assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking.

Predominant contribution of DGKζ over DGKα in the control of PKC/PDK-1-regulated functions in T cells.

Diacylglycerol kinase (DGK)-mediated consumption of the diacylglycerol (DAG) generated in response to antigen recognition is an important mechanism to limit T-cell function. Targeting DGK activity presents new opportunities for therapeutic manipulation of the immune response, but assessment of individual DGK functions is complex. T cells express two DGK isoforms, DGKα and DGKζ, and there are no isoform-specific inhibitors. Here we used short interfering RNA-mediated gene silencing in human T cells and DGKα- and DGKζ-deficient mice to define DGK isoform-specific regulation of key signaling pathways during T-cell activation. Our results identify DGKζ as the predominant brake on basal/tonic conditions as well as on downstream T-cell receptor/co-stimulatory signals. DGKζ silencing triggers basal RasGTP activation and facilitates enhanced membrane stability of protein kinase C alpha as well as increased activity of AGC kinases. Downstream of T-cell receptor/co-stimulation, DGKζ silencing results in enhanced and maintained recruitment of PKC theta to the membrane, as well as phosphoinositide-dependent protein kinase-1 activation and scaffolding functions. Our studies identify a previously unrecognized DGKζ contribution as a negative regulator of the crosstalk between phospholipase C-gamma- and phosphoinositide 3-kinase-regulated pathways. This DGKζ input helps to explain previous observations in DGK-deficient mice and suggests that the development of isoform-specific DGK inhibitors is of great interest for the manipulation of distinct aspects of T-cell responses.

Shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling.

Diacylglycerol signals by binding and activating C1 domain-containing proteins expressed principally in neuronal and immune tissues. This restricted expression profile suggests that diacylglycerol-regulated signals are particularly relevant in cell-cell communication processes in which active endocytosis and exocytosis take place. Not surprisingly, various experimental approaches have demonstrated a crucial role for diacylglycerol effectors and metabolizing enzymes in the control of immune responses, neuron communication and phagocytosis. Current research delineates a scenario in which coordinated decoding of diacylglycerol signals is translated into complex biological responses such as neuronal plasticity, T cell development or cytolytic killing. Diacylglycerol functions reach maximal diversity in these highly specialized systems in which signal intensity directly regulates distinct biological outcomes. This review brings together the most recent studies, emphasizing the contribution of compartmentalized DAG metabolism to orientated signaling events.

Transient PKCα shuttling to the immunological synapse is governed by DGKζ and regulates L-selectin shedding.

Considerable evidence indicates that diacylglycerol (DAG) generation at the immunological synapse (IS) determines T cell functions by regulating the duration and amplitude of Ras/ERK signals. The exact mechanism by which DAG regulates Ras/ERK activation downstream of the T cell receptor (TCR) nonetheless remains poorly understood. Here we characterize PKCα as a previously unrecognized component of the machinery that translates cell receptor occupancy into Ras/ERK-propagated signals. We show transient translocation of PKCα to the IS, mediated by DAG generation at the contact area. Diacylglycerol kinase (DGK)ζ negatively regulated PKCα translocation kinetics, whereas PKCα activity limited its own persistence at the IS. Coordinated activation of DGKζ and PKCα in response to antigen recognition regulated the amplitude and duration of Ras/ERK activation; this in turn mediated early processes of T cell surface proteolysis such as L-selectin shedding. Analysis of DGKζ-deficient mice further showed that increased DAG signaling is translated to downstream elements of this pathway, as reflected by enhanced PKCα-dependent L-selectin shedding. We propose that early activation of a DAG-PKCα axis contributes to the mechanisms by which antigen affinity translates into TCR biological responses.

Diacylglycerol kinases: A look into the future of immunotherapy.

Cancer still represents the second leading cause of death right after cardiovascular diseases. According to the World Health Organization (WHO), cancer provoked around 10 million deaths in 2020, with lung and colon tumors accounting for the deadliest forms of cancer. As tumor cells become resistant to traditional therapeutic approaches, immunotherapy has emerged as a novel strategy for tumor control. T lymphocytes are key players in immune responses against tumors. Immunosurveillance allows identification, targeting and later killing of cancerous cells. Nevertheless, tumors evolve through different strategies to evade the immune response and spread in a process called metastasis. The ineffectiveness of traditional strategies to control tumor growth and expansion has led to novel approaches considering modulation of T cell activation and effector functions. Program death receptor 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) showed promising results in the early 90s and nowadays are still being exploited together with other drugs for several cancer types. Other negative regulators of T cell activation are diacylglycerol kinases (DGKs) a family of enzymes that catalyze the conversion of diacylglycerol (DAG) into phosphatidic acid (PA). In T cells, DGKα and DGKζ limit the PLCγ/Ras/ERK axis thus attenuating DAG mediated signaling and T cell effector functions. Upregulation of either of both isoforms results in impaired Ras activation and anergy induction, whereas germline knockdown mice showed enhanced antitumor properties and more effective immune responses against pathogens. Here we review the mechanisms used by DGKs to ameliorate T cell activation and how inhibition could be used to reinvigorate T cell functions in cancer context. A better knowledge of the molecular mechanisms involved upon T cell activation will help to improve current therapies with DAG promoting agents.

Sorting Nexin 27-dependent regulation of Lck and CD4 tunes the initial stages of T-cell activation.

Sorting nexin (SNX) 27 is a unique member of the SNX family of proteins that mediates the endosome-to-plasma membrane trafficking of cargos bearing a PSD95/Dlg1/ZO-1 (PDZ)-binding motif. In brain, SNX27 regulates synaptic plasticity, and its dysregulation contributes to cognitive impairment and neuronal degeneration. In T lymphocytes, SNX27 partners with diacylglycerol (DAG) kinase ζ (DGKζ) to facilitate polarized traffic and signaling at the immune synapse (IS). By silencing SNX27 expression in a human T cell line, we demonstrate that SNX27 is a key regulator of the early T cell tyrosine-based signaling cascade. SNX27 transcriptionally controls CD4 abundance in resting conditions, and that of its associated molecule, Lck. This guarantees the adequate recruitment of Lck at the IS that is indispensable for subsequent activation of tyrosine phosphorylation regulated events. In contrast, reduced SNX27 expression enhances NFκB-dependent induction of CXCR4 and triggers production of lytic enzymes and pro-inflammatory cytokines. These results provide mechanistic explanation to previously described SNX27 function in the control of immune synapse organization and indicate that impaired SNX27 expression contributes to CD4 T cell dysfunction.

Modeling of host PDZ-dependent interactions with SARS-CoV-2 Envelope protein and changes in PDZ proteins expression in macrophages and dendritic cells.

PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins constitute a large family of scaffolds involved in a wide range of cellular tasks, and mainly studied in polarity functions. Diverse host PDZ proteins can be targeted by viral pathogens which express proteins containing PDZ-binding motifs (PDZbm). Previously, we have identified host PDZ-based interactions with the SARS-CoV-2 E protein (2E) in human monocytes. Here, we deepen the study of these interactions by docking and molecular dynamics analyses to identify the most favorable PDZ-PDZbm interaction of seven host PDZ proteins with the PDZbm of 2E. In addition, we analyzed changes in the expression of three of the PDZ proteins identified as 2E interactors in monocytes (syntenin, ZO-2, and IL-16), in human monocyte-derived macrophages (MΦ) and in dendritic cells (DCs) upon stimulation. Our results suggest that these PDZ proteins may have important functions in professional antigen-presenting cells (APCs), and their targeting by the PDZbm of 2E, a central virulence determinant of SARS-CoV-2, support the hypothesis that such PDZ-dependent interaction in immune cells may constitute a viral evasion mechanism. Inhibitor design based on the PDZbm of 2E in the development of drugs against a variety of diseases is discussed.

c-Abl tyrosine kinase regulates serum-induced nuclear export of diacylglycerol kinase α by phosphorylation at Tyr-218.

c-Abl is a tyrosine kinase involved in many cellular processes, including cell cycle control and proliferation. However, little is known about its substrates. Here, we show that c-Abl directly phosphorylates diacylglycerol kinase α (DGKα), an important regulator of many cellular events through its conversion of diacylglycerol to phosphatidic acid. We found that DGKα was transported from the cytoplasm to the nucleus in response to serum starvation, and serum restoration induced the nuclear export of the enzyme to the cytoplasm. This serum-induced export involves two tyrosine kinases, c-Src and c-Abl. The latter, c-Abl, is activated by c-Src, phosphorylates DGKα, and shuttles between the nucleus and the cytoplasm in a direction opposite to that of DGKα in response to serum restoration. Moreover, an in vitro phosphorylation assay using purified mutants of DGKα identified Tyr-218 as a site of phosphorylation by c-Abl. We confirmed these results for endogenous DGKα using an antibody specific for phospho-Tyr-218, and this phosphorylation was necessary for the serum-induced export of DGKα. These results demonstrate that the nucleo-cytoplasmic shuttling of DGKα is orchestrated by tyrosine phosphorylation by the Src-activated tyrosine kinase c-Abl and that this phosphorylation is important for regulating the function of cytoplasmic and/or nuclear DGKα.

Translocation dynamics of sorting nexin 27 in activated T cells.

Sorting nexin 27 (SNX27) belongs to the sorting nexin family of proteins, which participate in vesicular and protein trafficking. Similarly to all sorting nexin proteins, SNX27 has a functional PX domain that is important for endosome binding, but it is the only sorting nexin with a PDZ domain. We identified SNX27 as a partner of diacylglycerol kinase ζ (DGKζ), a negative regulator of T cell function that metabolises diacylglycerol to yield phosphatidic acid. SNX27 interacts with the DGKζ PDZ-binding motif in early/recycling endosomes in resting T cells; however, the dynamics and mechanisms underlying SNX27 subcellular localisation during T cell activation are unknown. We demonstrate that in T cells that encounter pulsed antigen-presenting cells, SNX27 in transit on early/recycling endosomes polarise to the immunological synapse. A fraction of SNX27 accumulates at the mature immunological synapse in a process that is dependent on vesicular trafficking, binding of the PX domain to phosphatidylinositol 3-phosphate and the presence of the PDZ region. Downmodulation of expression of either SNX27 or DGKζ results in enhanced basal and antigen-triggered ERK phosphorylation. These results identify SNX27 as a PDZ-containing component of the T cell immunological synapse, and demonstrate a role for this protein in the regulation of the Ras-ERK pathway, suggesting a functional relationship between SNX27 and DGKζ.

Diacylglycerol, when simplicity becomes complex.

Diacylglycerol (DAG) has unique functions as a basic component of membranes, an intermediate in lipid metabolism and a key element in lipid-mediated signaling. In eukaryotes, for example, impaired DAG generation and/or consumption have severe effects on organ development and cell growth associated with diseases such as cancer, diabetes, immune system disorders and Alzheimer's disease. Although DAG has been studied intensively as a signaling lipid, early models of its function are no longer adequate to explain its numerous roles. The interplay between enzymes that control DAG levels, the identification of families of DAG-regulated proteins, and the overlap among DAG metabolic and signaling processes are providing new interpretations of DAG function. Recent discoveries are also delineating the complex and strategic role of DAG in regulating biochemical networks.

Sorting Nexin 27 Enables MTOC and Secretory Machinery Translocation to the Immune Synapse.

Sorting nexin 27 (SNX27) association to the retromer complex mediates intracellular trafficking of cargoes containing PSD95/Dlg1/ZO-1 (PDZ)-binding C-terminal sequences from endosomes to the cell surface, preventing their lysosomal degradation. Antigen recognition by T lymphocyte leads to the formation of a highly organized structure named the immune synapse (IS), which ensures cell-cell communication and sustained T cell activation. At the neuronal synapse, SNX27 recycles PDZ-binding receptors and its defective expression is associated with synaptic dysfunction and cognitive impairment. In T lymphocytes, SNX27 was found localized at recycling endosomal compartments that polarized to the IS, suggesting a function in polarized traffic to this structure. Proteomic analysis of PDZ-SNX27 interactors during IS formation identify proteins with known functions in cytoskeletal reorganization and lipid regulation, such as diacylglycerol (DAG) kinase (DGK) ζ, as well as components of the retromer and WASH complex. In this study, we investigated the consequences of SNX27 deficiency in cytoskeletal reorganization during IS formation. Our analyses demonstrate that SNX27 controls the polarization towards the cell-cell interface of the PDZ-interacting cargoes DGKζ and the retromer subunit vacuolar protein sorting protein 26, among others. SNX27 silencing abolishes the formation of a DAG gradient at the IS and prevents re-localization of the dynactin complex component dynactin-1/p150Glued, two events that correlate with impaired microtubule organizing center translocation (MTOC). SNX27 silenced cells show marked alteration in cytoskeleton organization including a failure in the organization of the microtubule network and defects in actin clearance at the IS. Reduced SNX27 expression was also found to hinder the arrangement of signaling microclusters at the IS, as well as the polarization of the secretory machinery towards the antigen presenting cells. Our results broaden the knowledge of SNX27 function in T lymphocytes by showing a function in modulating IS organization through regulated trafficking of cargoes.