PP2Cδ Controls the Differentiation and Function of Dendritic Cells Through Regulating the NSD2/mTORC2/ACLY Pathway

Dendritic cells (DCs) are recognized as a key orchestrator of immune response and homeostasis, deregulation of which may lead to autoimmunity such as experimental autoimmune encephalomyelitis (EAE). Herein we show that the phosphatase PP2Cδ played a pivotal role in regulating DC activation and function, as PP2Cδ ablation caused aberrant maturation, activation, and Th1/Th17-priming of DCs, and hence induced onset of exacerbated EAE. Mechanistically, PP2Cδ restrained the expression of the essential subunit of mTORC2, Rictor, primarily through de-phosphorylating and proteasomal degradation of the methyltransferase NSD2 via CRL4DCAF2 E3 ligase. Loss of PP2Cδ in DCs accordingly sustained activation of the Rictor/mTORC2 pathway and boosted glycolytic and mitochondrial metabolism. Consequently, ATP-citrate lyse (ACLY) was increasingly activated and catalyzed acetyl-CoA for expression of the genes compatible with hyperactivated DCs under PP2Cδ deletion. Collectively, our findings demonstrate that PP2Cδ has an essential role in controlling DCs activation and function, which is critical for prevention of autoimmunity.

Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl– currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

C2CD6 is required for assembly of the CatSper calcium channel complex and fertilization

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here we report identification of an uncharacterized protein C2CD6 as a novel subunit of the CatSper ion channel complex. C2CD6 contains a calcium-dependent membrane targeting C2 domain. C2CD6 interacts with the CatSper calcium-selective core forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. Interestingly, transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm in vitro. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 may regulate CatSper assembly, target the CatSper complex to flagellar plasma membrane, and function as a calcium sensor. The identification of C2CD6 as an essential subunit may facilitate the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Rice FLOURY SHRUNKEN ENDOSPERM 5 Encodes a Putative Plant Organelle RNA Recognition Protein that Is Required for cis-Splicing of Mitochondrial nad4 Intron 1

Background The sequences of several important mitochondrion-encoded genes involved in respiration in higher plants are interrupted by introns. Many nuclear-encoded factors are involved in splicing these introns, but the mechanisms underlying this splicing remain unknown. Results We isolated and characterized a rice mutant named floury shrunken endosperm 5 (fse5). In addition to having floury shrunken endosperm, the fse5 seeds either failed to germinate or produced seedlings which grew slowly and died ultimately. Fse5 encodes a putative plant organelle RNA recognition (PORR) protein targeted to mitochondria. Mutation of Fse5 hindered the splicing of the first intron of nad4, which encodes an essential subunit of mitochondrial NADH dehydrogenase complex I. The assembly and NADH dehydrogenase activity of complex I were subsequently disrupted by this mutation, and the structure of the mitochondria was abnormal in the fse5 mutant. The FSE5 protein was shown to interact with mitochondrial intron splicing factor 68 (MISF68), which is also a splicing factor for nad4 intron 1 identified previously via yeast two-hybrid (Y2H) assays. Conclusion Fse5 which encodes a PORR domain-containing protein, is essential for the splicing of nad4 intron 1, and loss of Fse5 function affects seed development and seedling growth.

Differentially accessible Cdc4 phospho-degrons regulate Ctf19CCAN kinetochore subunit stability in mitosis

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. How effective, yet strictly centromere-dependent kinetochore assembly is coupled to cell cycle progression is incompletely understood. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-Phase and targets the protein for degradation. Binding of the Mtw1 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature-sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic to cells, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits protects against ectopic kinetochore assembly and contributes to mitotic checkpoint silencing. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

FOXP3 and Tip60 Structural Interactions Relevant to IPEX Development Lead to Potential Therapeutics to Increase FOXP3 Dependent Suppressor T Cell Functions

Regulatory T (Treg) cells play a role in the maintenance of immune homeostasis and are critical mediators of immune tolerance. The Forkhead box P3 (FOXP3) protein acts as a regulator for Treg development and function. Mutations in the FOXP3 gene can lead to autoimmune diseases such as Immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome in humans, often resulting in death within the first 2 years of life and a scurfy like phenotype in Foxp3 mutant mice. We discuss biochemical features of the FOXP3 ensemble including its regulation at various levels (epigenetic, transcriptional, and post-translational modifications) and molecular functions. The studies also highlight the interactions of FOXP3 and Tat-interacting protein 60 (Tip60), a principal histone acetylase enzyme that acetylates FOXP3 and functions as an essential subunit of the FOXP3 repression ensemble complex. Lastly, we have emphasized the role of allosteric modifiers that help stabilize FOXP3:Tip60 interactions and discuss targeting this interaction for the therapeutic manipulation of Treg activity.

Role of POT1 in Human Cancer

Telomere abnormalities facilitate cancer development by contributing to genomic instability and cellular immortalization. The Protection of Telomeres 1 (POT1) protein is an essential subunit of the shelterin telomere binding complex. It directly binds to single-stranded telomeric DNA, protecting chromosomal ends from an inappropriate DNA damage response, and plays a role in telomere length regulation. Alterations of POT1 have been detected in a range of cancers. Here, we review the biological functions of POT1, the prevalence of POT1 germline and somatic mutations across cancer predisposition syndromes and tumor types, and the dysregulation of POT1 expression in cancers. We propose a framework for understanding how POT1 abnormalities may contribute to oncogenesis in different cell types. Finally, we summarize the clinical implications of POT1 alterations in the germline and in cancer, and possible approaches for the development of targeted cancer therapies.

Late Adolescence Mortality in Mice with Brain-Specific Deletion of the Volume-Regulated Anion Channel Subunit LRRC8A

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is indispensable for cell volume regulation but its broader physiological functions remain under investigation. Astrocyte-targeted Lrrc8a deletion in the nervous system reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against ischemic damage. Here we show that deletion of LRRC8A in all brain cells, using NestinCre-driven Lrrc8afl/fl excision, is lethal. Mice devoid of brain LRRC8A are born close to the expected Mendelian ratio and develop without overt histological abnormalities. Nevertheless, they all die between 5 to 8 weeks of age with a seizure-like phenotype. Consistent with seizures, we found disruptions in cell excitability, GABAergic signaling, and astrocytic production of the GABA precursor glutamine, all of which might contribute to mortality. This work provides the first evidence of a critical role for VRAC in control of brain excitability during maturation.

Loss of Hem1 disrupts macrophage function and impacts on migration, phagocytosis and integrin-mediated adhesion

ABSTRACTThe hematopoietic-specific protein 1 (Hem1) comprises an essential subunit of the WAVE Regulatory Complex (WRC) in immune cells. WRC has a fundamental role in Arp2/3 complex activation and the protrusion of branched actin networks in motile cells.Hem1 deficiency leads to suppression of the entire WRC in immune cells. Defective WRC function in macrophages results in loss of lamellipodia and migration defects. Moreover, phagocytosis, commonly accompanied by lamellipodium protrusion during cup formation, is altered in Hem1 null cells concerning frequency and efficacy. When analyzing cell spreading, adhesion and podosome formation, we found that Hem1 null cells are capable, in principle, of podosome formation and consequently, do not show any quantitative differences in extracellular matrix degradation. Their adhesive behavior, however, was significantly altered. Specifically, adhesion as well as de-adhesion of Hem1 null cells was strongly compromised, likely contributing to the observed reduced efficiency of phagocytosis. In line with this, phosphorylation of the prominent adhesion component paxillin was diminished. Non-hematopoietic somatic cells disrupted in expression for both Hem1 and its ubiquitous orthologue Nck-associated protein 1 (Nap1) or the essential WRC components Sra-1/PIR121 did not only confirm defective paxillin phosphorylation, but also revealed that paxillin turnover in focal adhesions is accelerated in the absence of WRC. Finally, adhesion assays using platelets lacking functional WRC as model system unmasked radically decreased αIIbβ3 integrin activation.Our results thus demonstrate that WRC-driven actin networks impact on integrin-dependent processes controlling formation and dismantling of different types of cell-substratum adhesion.One sentence summaryInterference of Hem1 function in mice and cells uncovers a hitherto unrecognized role in integrin-mediated cell adhesion that is crucial for macrophage function and connects to recently discovered immunodeficiencies in patients carrying Hem1 mutations.