Cytoplasmic Tail Of CD4 Research Articles

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery—it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket, and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of DMSO and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

Semisynthesis of segmentally isotope-labeled and site-specifically palmitoylated CD44 cytoplasmic tail

CD44, a ubiquitously expressed transmembrane receptor, plays a crucial role in cell growth, migration, and tumor progression. Dimerization of CD44 is a key event in signal transduction and has emerged as a potential target for anti-tumor therapies. Palmitoylation, a posttranslational modification, disrupts CD44 dimerization and promotes CD44 accumulation in ordered membrane domains. However, the effects of palmitoylation on the structure and dynamics of CD44 at atomic resolution remain poorly understood. Here, we present a semisynthetic approach combining solid-phase peptide synthesis, recombinant expression, and native chemical ligation to investigate the impact of palmitoylation on the cytoplasmic domain (residues 669-742) of CD44 (CD44ct) by NMR spectroscopy. A segmentally isotope-labeled and site-specifically palmitoylated CD44 variant enabled NMR studies, which revealed chemical shift perturbations and indicated local and long-range conformational changes induced by palmitoylation. The long-range effects suggest altered intramolecular interactions and potential modulation of membrane association patterns. Semisynthetic, palmitoylated CD44ct serves as the basis for studying CD44 clustering, conformational changes, and localization within lipid rafts, and could be used to investigate its role as a tumor suppressor and to explore its therapeutic potential.

CD4 and LAG-3 from sharks to humans: related molecules with motifs for opposing functions.

CD4 and LAG-3 are related molecules that are receptors for MHC class II molecules. Their major functional differences are situated in their cytoplasmic tails, in which CD4 has an activation motif and LAG-3 an inhibitory motif. Here, we identify shark LAG-3 and show that a previously identified shark CD4-like gene has a genomic location, expression pattern, and motifs similar to CD4 in other vertebrates. In nurse shark (Ginglymostoma cirratum) and cloudy catshark (Scyliorhinus torazame), the highest CD4 expression was consistently found in the thymus whereas such was not the case for LAG-3. Throughout jawed vertebrates, the CD4 cytoplasmic tail possesses a Cx(C/H) motif for binding kinase LCK, and the LAG-3 cytoplasmic tail possesses (F/Y)xxL(D/E) including the previously determined FxxL inhibitory motif resembling an immunoreceptor tyrosine-based inhibition motif (ITIM). On the other hand, the acidic end of the mammalian LAG-3 cytoplasmic tail, which is believed to have an inhibitory function as well, was acquired later in evolution. The present study also identified CD4-1, CD4-2, and LAG-3 in the primitive ray-finned fishes bichirs, sturgeons, and gars, and experimentally determined these sequences for sterlet sturgeon (Acipenser ruthenus). Therefore, with CD4-1 and CD4-2 already known in teleosts (modern ray-finned fish), these two CD4 lineages have now been found within all major clades of ray-finned fish. Although different from each other, the cytoplasmic tails of ray-finned fish CD4-1 and chondrichthyan CD4 not only contain the Cx(C/H) motif but also an additional highly conserved motif which we expect to confer a function. Thus, although restricted to some species and gene copies, in evolution both CD4 and LAG-3 molecules appear to have acquired functional motifs besides their canonical Cx(C/H) and ITIM-like motifs, respectively. The presence of CD4 and LAG-3 molecules with seemingly opposing functions from the level of sharks, the oldest living vertebrates with a human-like adaptive immune system, underlines their importance for the jawed vertebrate immune system. It also emphasizes the general need of the immune system to always find a balance, leading to trade-offs, between activating and inhibiting processes.

Discovery of FERM domain protein–protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer’s disease

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Temperature as an Extra Dimension in Multidimensional Protein NMR Spectroscopy.

NMR spectroscopy is a particularly informative method for studying protein structures and dynamics in solution; however, it is also one of the most time-consuming. Modern approaches to biomolecular NMR spectroscopy are based on lengthy multidimensional experiments, the duration of which grows exponentially with the number of dimensions. The experimental time may even be several days in the case of 3D and 4D spectra. Moreover, the experiment often has to be repeated under several different conditions, for example, to measure the temperature-dependent effects in a spectrum (temperature coefficients (TCs)). Herein, a new approach that involves joint sampling of indirect evolution times and temperature is proposed. This allows TCs to be measured through 3D spectra in even less time than that needed to acquire a single spectrum by using the conventional approach. Two signal processing methods that are complementary, in terms of sensitivity and resolution, 1) dividing data into overlapping subsets followed by compressed sensing reconstruction, and 2) treating the complete data set with a variant of the Radon transform, are proposed. The temperature-swept 3D HNCO spectra of two intrinsically disordered proteins, osteopontin and CD44 cytoplasmic tail, show that this new approach makes it possible to determine TCs and their non-linearities effectively. Non-linearities, which indicate the presence of a compact state, are particularly interesting. The complete package of data acquisition and processing software for this new approach are provided.

Temperature as an extra dimension in multidimensional protein NMR.

Nuclear magnetic resonance spectroscopy (NMR) is a particularly informative method for studying protein structure and dynamics in solution; however, it is also one of the most time-consuming. Modern approaches to biomolecular NMR are based on lengthy multidimensional (ND) experiments whose duration grows exponentially with the number of dimensions. The experimental time may even be several days in the case of 3D and 4D spectra. Moreover, the experiment often has to be repeated in several different conditions, for example in order to measure the temperature-dependent effects in a spectrum (temperature coefficients, TCs). In this paper we propose a new approach that involves joint sampling of indirect evolution times and temperature. This allows us to measure TCs with 3D spectra in even less time than is needed to acquire a single spectrum using the conventional approach. We propose two signal processing methods that are complementary in terms of sensitivity and resolution: a) dividing the data into overlapping subsets followed by compressed sensing reconstruction, and b) treating the complete data set with a variant of the Radon transform. The temperature-swept 3D HNCO spectra of two intrinsically disordered proteins---osteopontin and CD44 cytoplasmic tail---show that our new approach makes it possible to determine TCs and their non-linearities effectively. Non-linearities, which indicate the presence of a compact state, are particularly interesting. We provide the complete package of data acquisition and processing software for this new approach.

Distinct CD28 signaling requirements determine the alternative fates of thymic agonist selection

Abstract During thymic T cell development, T cells strongly reactive to self-Ag are eliminated from the conventional T cell repertoire by clonal deletion. Alternatively, however, a number of T cell subsets are positively selected by strong recognition of self-Ag/MHC by TCR in thymus, termed agonist selection. Those T cell subsets include 1) Foxp3+ Treg cells, 2) invariant NKT (iNKT) cells, and 3) αβTCR+ PD-1+ CD4− CD8− precursors of CD8αα IELs (pIEL). In addition to a strong TCR signal, B7-CD28 co-stimulation has a role in these agonist selections. This role is context dependent. For instance, B7-CD28 has a positive role for thymic Treg and iNKT cell generation, while it has a negative role for thymic pIEL generation. These differences may indicate involvement of distinct CD28 signaling in these events. To test this hypothesis, we analyzed CD28 cytoplasmic tail mutant mice in which tyrosine 170 residue was substituted to phenylalanine (Y170F), proline residues in PYAP motif were substituted to alanine (AYAA), or both mutation were present (Y170F/AYAA). Generation of self-Ag-specific Treg, defined by pMHCII tetramer binding, was attenuated in AYAA mice but not in Y170F mice, consistent with the reported role of the PYAP motif for total Treg generation. Meanwhile, interestingly, the negative role of CD28 for self-Ag-specific pIEL generation was not affected by Y170F and/or AYAA substitution, indicating that this CD28 effect was mediated by motif(s) other than Y170 and PYAP. Analysis of thymic iNKT cell (NKT1, 2 and 17) development in CD28 mutant mice is currently in progress. These results suggest that the differing roles of B7-CD28 costimulation for agonist selection fates are mediated via qualitatively distinct CD28 signaling.

Gamma secretase dependent release of the CD44 cytoplasmic tail upregulates IFI16 in cd44-/- tumor cells, MEFs and macrophages.

The adhesion molecule and co-receptor of receptor tyrosine kinases, CD44, is expressed in all cells of the immune system, but also in numerous non-immune cells. CD44 plays roles in the cellular response to different pathogens. The molecular actions of CD44 during these processes are by and large still unknown. The CD44 molecule undergoes a sequential proteolytic cleavage which leads to the release of a soluble intracellular domain (CD44-ICD). Previous reports had shown that the CD44-ICD is taken up into the nucleus where it enhances transcription of specific target genes. By RNA profiling we identified a CD44-dependent transcriptional increase of interferon-responsive genes, among them IFI16. IFI16 is important in the innate immune response. It senses and binds pathogenic DNA and, together with cGAS, activates the cGAS-cGAMP-STING pathway and induces the expression of genes relevant for the response, e.g. IFN-β. Our results show that the enhancement of IFI16 expression depended on CD44 cleavage. A CD44-negative tumor cell line, embryonic fibroblasts and bone marrow-derived macrophages from cd44-/- mice were reduced in their response to IFN-γ, to viral DNA fragments and to Listeria monocytogenes infection. We could rescue the deficiency of CD44 negative RPM-MC cells and cd44-/- MEFs by expressing only the soluble CD44-ICD in the absence of any other CD44 domain. Expression of the CD44-ICD carrying a mutation that prevented the uptake into the nucleus, could not rescue the absence of CD44. This molecular aspect of regulation by CD44 may explain part of the immune phenotypes of mice with cd44 gene disruption.

1H, 15N, 13C resonance assignment of the human CD44 cytoplasmic tail (669\u2013742)

CD44 is a universally and abundantly expressed single-pass type I protein that spans the cytoplasmic membrane and is considered the principal receptor for hyaluronan in the extracellular matrix. CD44 exerts a multitude of biological functions, especially in cell adhesion and migration, and its deregulation has several pathological implications, including a putative role in cancer cell dissemination. Here we report the NMR chemical shift assignment of the recombinant intrinsically disordered CD44 cytoplasmic region (669–742).

CD28 Induces Mitochondrial Respiration Dependent Reactive Oxygen Species (ROS) Signaling for Metabolic Fitness and Survival in Long-Lived Plasma Cells

Abstract Durable humoral immunity depends upon bone marrow resident long-lived plasma cell (LLPC) survival. We have shown that CD28, the canonical T-cell costimulatory molecule, is required for LLPC survival; however, the mechanistic basis for this is unclear. We have published that of two binding motifs in the CD28 cytoplasmic tail, only the Grb2/Vav moiety, and not the direct PI3k binding motif, supports CD28-mediated survival. CD28 is known to induce glycolysis in T-cells. In contrast, CD28 activation in both LLPCs and human PCs induces glucose metabolism through mitochondrial respiration. A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). Interestingly, the mitochondrial respiration-dependent production of ROS is required for CD28-mediated survival. Previously published data demonstrate the ability of ROS to signal through both NFkB and PI3k. CD28-induced ROS signal through PI3k in LLPCs for increased glucose uptake and the inhibition of PI3k prevents CD28-mediated survival. The ROS-PI3k signal drives the phosphorylation of both S6k and rpS6, downstream targets of mTORC1, and this regulation is independent of Blimp1 expression. Irf4, a critical PC survival factor, is upregulated by CD28-dependent NFkB activation. To our great surprise, ROS inhibition prevented the induction of mitochondrial respiration itself, demonstrating that signals emanating downstream of ROS govern the metabolic program for survival in LLPCs. Taken together, these data suggest a model wherein CD28 signals directly through Grb2/Vav to induce mitochondrial respiration and ROS production. ROS then reinforce signaling through PI3k and NFkB to increase Irf4 expression and metabolic fitness for LLPC survival.

RIP1 has a role in CD40-mediated apoptosis in human follicular lymphoma cells

CD40 is a cell surface receptor which belongs to tumor necrosis factor receptor (TNFR) family members. It transmits signals that regulate diverse cellular responses such as proliferation, differentiation, adhesion molecule expression and apoptosis. Unlike other TNFR family members (TRAIL-R, Fas-R and TNFR1), the CD40 cytoplasmic tail lacks death domain. However, CD40 is capable of inducing apoptosis in different types of cancer cells including lymphoma. The apoptotic effect of CD40 is linked to the involvement of Fas, TRAIL or receptor interacting protein 1 (RIP1) kinase. We have previously shown that CD40 activation has anti-apoptotic or apoptotic effect in follicular lymphoma (FL) cell lines. In this study, we investigated the mechanism by which CD40 mediates apoptosis in a follicular lymphoma cell line, HF4.9. We show here that CD40-induced apoptosis was dependent on caspase-8 activation because caspase-8 specific inhibitor, Z-IETD-FMK completely prevented apoptosis. Therefore, the involvement of TRAIL, Fas and RIP1 in caspase-8 activation was examined. The exogenous TRAIL-induced apoptosis was fully prevented by anti-TRAIL neutralizing antibody. However, the antibody had no effect on CD40-induced apoptosis indicating that CD40 did not induce the expression of endogenous TRAIL in HF4.9 cells. Moreover, the cells were not sensitive to Fas-mediated apoptosis. Interestingly, RIP1 specific inhibitor, necrostatin-1 decreased CD40-induced apoptosis, which showed that RIP1 has a role in caspase-8 activation. In conclusion, the survival or apoptotic effects of CD40-mediated signaling might be related to the differentiation stages of FL cells.

CD28 induces survival in long-lived plasma cells through NFκB mediated Irf4 upregulation and mitochondrial respiration-dependent reactive oxygen species production.

Abstract Durable humoral immunity is dependent upon sustained antibody production by long-lived plasma cells (LLPCs). We have published that CD28 is required for LLPC survival and antibody production in vivo; however, the mechanistic basis for this is unclear. In T cells, CD28 is known to induce glycolysis at the expense of respiration. Surprisingly, CD28 induces mitochondrial respiration and biogenesis in LLPCs while also increasing glucose uptake. Paradoxically, respiration-dependent reactive oxygen species (ROS) are required for CD28-mediated survival. Using knock-in mice with mutations in the CD28 cytoplasmic tail, we demonstrate that CD28-Grb2-Vav-Slp76 signaling is required for the survival of LLPCs with high mitochondrial mass and ROS in vivo. Mechanistically, CD28 induces Irf4 in an NFκB-dependent manner. CD28-ROS drive cRel translocation, an NFκB subunit, which can bind directly to the Irf4 promoter. We also describe a super enhancer element further upstream of Irf4 driven by the NFκB subunit p65, induced by CD28 specifically in LLPCs. Furthermore, inhibition of NFκB abrogates CD28-mediated glucose uptake, mitochondrial respiration and biogenesis. Irf4 heterozygous LLPCs have lower mitochondrial mass in vivo, and knock-down of Irf4 decreases the mitochondrial mass. This leads to a model wherein CD28, through Grb2-Vav-Slp-76, reinforces LLPC survival through the induction of mitochondrial biogenesis and respiration. Respiration-dependent ROS then augment NFκB-mediated Irf4 expression through direct promoter activity and a previously undescribed super enhancer element. CD28, therefore, makes an attractive target for the augmentation of vaccine design and in the alleviation of antibody-mediated autoimmunity.

CD28 Induces Mitochondrial Respiration through Irf4 for Long Lived Plasma Cells Survival

Successful vaccination strategies against emerging pathogens like that of the Zika virus depend upon the sustained production of antigen-specific antibodies by terminally differentiated B cells known as plasma cells. Because the half-life of circulating antibodies is on the order of days to weeks, durable humoral immunity requires the induction of long-lived plasma cells (LLPCs) that home to specialized survival niches in the bone marrow and live indefinitely. Therefore, understanding the cellular and molecular interactions that govern LLPC survival are critical for the design of effective vaccines.We have published that CD28, the canonical T cell co-stimulatory molecule, is required for LLPC survival in the bone marrow. In order to maintain this extraordinary degree of longevity, LLPCs must be able to compete successfully in an environment wherein normal hematopoiesis puts constraints on the availability of nutrients. Furthermore, LLPCs are highly biosynthetic as antibody producing factories and necessitate a high level of metabolic activity. In T cells CD28 is known to induce glycolysis at the expense of mitochondrial respiration. To our great surprise, CD28 signaling in LLPCs increases mitochondrial respiration whilst not greatly affecting glycolysis. However, CD28 enhanced the glycolytic capacity and reserve, suggesting that CD28 signaling regulates their ability to better compete for nutrient availability and metabolic fitness. CD28 induces the expression of the glucose transporter Glut1 and subsequent glucose uptake as assessed by staining with the autofluorescent glucose analogue NBDG. In contrast to the T cell literature, CD28 increases the mitochondrial mass and membrane potential in LLPCs, suggesting that rather than using glucose primarily for glycolysis it is converted into pyruvate which then is used in mitochondrial respiration.Mechanistically, CD28 induces NFkB dependent IRf4 upregulation, a transcription factor known to regulate metabolism in T cells and survival in LLPCs. Furthermore, inhibition of NFkB abrogates CD28-induced increases in glucose uptake and mitochondrial mass. We have recently published that the Grb2/Vav binding domain on the CD28 cytoplasmic tail is required for CD28-mediated survival. In mice wherein this domain is mutated (AYAA mutants), LLPCs have decreased glucose uptake and mitochondrial mass. Plasma cells from AYAA mutant mice also have decreased levels of Irf4, facilitating a model wherein CD28 through its Grb2/Vav binding domain increased Irf4 for metabolic fitness. We also describe for the first time an NFkB superenhancer element upstream of the Irf4 transcriptional start site, suggesting that CD28 may govern both direct Irf4 promoter activity as well as DNA folding for increased Irf4 production. By using siRNA to target Irf4 at the genetic level, we see diminished mitochondrial mass, suggesting that Irf4 is the direct regulator of CD28-regulated metabolism in LLPCs.A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). CD28 increased the amount of ROS production in LLPCs as seen by staining with a mitochondrial-ROS specific dye. This is counterintuitive, as ROS are well characterized cell damaging agents. Paradoxically, when we inhibited ROS with MnTBAP, a ROS scavenger, CD28 no longer conferred a survival signal in LLPCs. Taken together these results suggest a model wherein CD28 through its Grb2/Vav binding domain induces NFkB dependent upregulation of Irf4 directly on the promoter and augments further Irf4 production via a previously undescribed superenhancer element upstream of the Irf4 gene. Irf4 then goes on to increase mitochondrial respiration for CD28-mediated LLPC survival and metabolic fitness. This understanding will not only allow for the augmentation of vaccine design and efficacy, but may also lead to novel therapeutic strategies to combat antibody-mediated autoimmunity. DisclosuresNo relevant conflicts of interest to declare.

CD28 Promotes Plasma Cell Survival, Sustained Antibody Responses, and BLIMP-1 Upregulation through Its Distal PYAP Proline Motif.

In health, long-lived plasma cells (LLPC) are essential for durable protective humoral immunity, and, conversely, in disease are a major source of pathogenic Abs in autoimmunity, graft rejection, and allergy. However, the molecular basis for their longevity is largely unknown. We have recently found that CD28 signaling in plasma cells (PC) is essential for sustaining Ab titers, by supporting the survival of LLPC, but not short-lived PC (SLPC). We now find that, unlike SLPC, CD28 activation in LLPC induces prosurvival downstream Vav signaling. Knockin mice with CD28 cytoplasmic tail mutations that abrogate Vav signaling (CD28-AYAA) had significantly fewer LLPC but unaffected SLPC numbers, whereas mice with mutations that abrogate PI3K signaling (CD28-Y170F) were indistinguishable from wild-type controls. This was consistent with the loss of CD28's prosurvival effect in LLPC from CD28-AYAA, but not CD28-Y170F, mice. Furthermore, the CD28 Vav motif in the B lineage was essential for the long-term maintenance of Ag-specific LLPC populations and Ab titers in vivo. Signaling downstream of the CD28 Vav motif induced previously undescribed transcriptional regulation of B lymphocyte-induced maturation protein-1, a key mediator of PC differentiation and maintenance. These findings suggest CD28 signaling in LLPC modulates the central B lymphocyte-induced maturation protein-1 transcriptional nexus involved in long-term survival and function.

CD44 regulates cell proliferation, migration, and invasion via modulation of c-Src transcription in human breast cancer cells

CD44 was recently identified as a cancer initiation marker on the cell membrane. The cytoplasmic tail of CD44 is known to bind ERM (ezrin, radixin, moesin) proteins, cytoskeletal proteins like ankyrin, and the non-receptor tyrosine kinase c-Src. CD44 transmits its oncogenic signaling via c-Src and its downstream effectors. To investigate the role of CD44 in breast cancer cells, we generated CD44 knock-down cells via retroviral delivery of shRNA against CD44. We found that silencing of CD44 decreased the proliferation, migration, and invasion of breast cancer cells. The expression and activity of cell migration-related proteins, including c-Src, paxillin, and FAK were decreased by CD44 silencing. We also found that the c-Jun protein level was negatively regulated via induction of a GSK-3β-dependent degradation pathway in CD44 knock-down cells. The expression level of Sp1, a target gene product of c-Jun, was also decreased in these cells. Finally, CD44 knock-down suppressed both mRNA and protein levels of c-Src and its downstream MAPK pathway as a result of down-regulation of Sp1 as a transcription factor for c-Src. Collectively, these results indicate that biological changes induced by CD44 silencing are mediated by cumulative down-regulation of c-Jun, Sp1, and c-Src in human breast cancer cells.

Defects in regulatory T cells due to CD28 deficiency induce a qualitative change of allogeneic immune response in chronic graft-versus-host disease.

In patients receiving allogeneic hematopoietic cell transplantation, chronic graft-versus-host disease (cGVHD) remains a frequent complication and resembles autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. Our previous work demonstrated the critical role of CD28 costimulation of donor T cells for GVHD induction. In this study, we investigate the role of CD28 costimulation of host T cells in cGVHD. CD28-intact mice as hosts showed systemic lupus erythematosus-type cGVHD, whereas CD28-deficient mice developed a distinct phenotype of cGVHD, with fibrotic damage in skin and internal organs, resembling systemic sclerosis. This phenotype was due to a lack of signaling through the C-terminal proline-rich motif within host CD28's cytoplasmic tail, a motif previously shown to be required for development of regulatory T cells (Tregs) and function of conventional T cells. Adoptive transfer experiments demonstrated that a defect in host CD4(+)CD25(+) Tregs, but not in conventional T cells, was responsible for disease phenotype. Host Treg deficiency altered the cytokine pattern of donor CD4(+) T cells and the Ag specificity of autoantibodies, and these might lead to phenotypic change. Thus, host CD28 signaling controlled the pathogenesis of cGVHD through effects on host Tregs, whose status impacts qualitatively on the allogeneic immune responses.

Phosphatidylinositol 4,5‐bisphosphate Activates the Cell Adhesion Molecule CD44 and Assembles a CD44‐Ezrin Heterotetramer Complex as Revealed by Small Angle neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell‐cell and cell‐matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co‐localizes with the membrane‐cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an auto‐inhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5‐biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin. Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero‐tetramer complex of CD44ct with Ezrin. This study reveals a novel auto‐regulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct‐Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2‐CD44‐Ezrin complexes.

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.

CD28 Mutations in Peripheral T-Cell Lymphomagenesis and Progression

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of diseases with poor prognosis and no standard therapy due to widely varying responses to treatments, thus new therapeutic targets need to be identified. Gene expression profiling (GEP) has helped to separate classes of these diseases to predict survival. Recent genetic studies on the two most common subtypes of PTCL in the Western world, angioimmunoblastic T-cell lymphoma (AITL) and PTCL, not otherwise specified (PTCL-NOS), have revealed recurrent mutations in the epigenetic modifiers TET2, IDH2, and DNMT3A; in the small GTPase RHOA; and rarely in the T-cell receptor (TCR) adaptor protein FYN. Because TCR stimulation is necessary for normal T-cell expansion, activating mutations in the stimulation (CD3/TCR) and costimulation (CD28) pathways could be involved in malignant lymphopoiesis.We performed whole-transcriptome sequencing on a small set of primary PTCL cases and found mutations in CD28, including an aspartate 124 mutation within a fusion transcript between CD28 and family member ICOS as well as a threonine 195 mutation. On targeted re-sequencing of 92 PTCL cases, we found two AITL cases and one ALK- anaplastic large cell lymphoma case with an aspartate 124 mutation (variant frequencies [VF]: 1.03% to 5.90%). We also found mutations at threonine 195 (two AITL, one PTCL-NOS, Tbx21 subtype; VF: 2.90% to 12.30%). Additionally, we found two recurrent mutations with low variant frequencies (<2.0%). We also determined each targeted sequencing sample’s RHOA mutation status; high-VF CD28 mutations also had RHOA glycine 17 mutations, almost all of which were to valine (G17V) consistent with previous reports of RHOA mutations in PTCLs.Surface Plasmon Resonance (SPR) analysis of the two highest-VF mutated residues D124 and T195 in CD28 show increased affinities of CD28 mutants for partners. The D124 to valine mutation (D124V) within the extracellular ligand binding region increases affinity for CD86. The T195 to proline mutation (T195P) increases binding of GRB2 and GADS/GRAP2 to the CD28 cytoplasmic tail at the intracellular SH2-binding domain. Molecular modeling of these mutations offers possible explanations for these differential affinities, including potential increased electrostatic binding at the extracellular interface and binding partner side-chain stabilization intracellularly. Further, these mutations have a significantly increased capacity to activate NF-κB upon ligation of CD80 or CD86 and anti-CD3 stimulation.Mutations in CD28 often occur with low frequency in PTCL tumor samples; given this low frequency, the mutations are likely not a primary mechanism of lymphomagenesis. It is possible that over-activation of CD28 represents a late-stage event in clonal evolution, allowing a tumor subclone to further grow and proliferate. These CD28-mutant clones may play a role in later disease course, in refractory disease, or in post-therapeutic relapse. DisclosuresFu:Nanostring: The author is a potential inventor on a patent application using Nanostring technology for the Lymph2Cx assay, which has been licensed from the NIH by Nanostring Patents & Royalties.

Structural basis of DDB1-and-Cullin 4-associated Factor 1 (DCAF1) recognition by merlin/NF2 and its implication in tumorigenesis by CD44-mediated inhibition of merlin suppression of DCAF1 function.

Merlin, a tumor suppressor encoded by the neurofibromatosis type 2 gene, has been shown to suppress tumorigenesis by inhibiting the Cullin 4-RING E3 ubiquitin ligase CRL4(DCAF) (1) in the nucleus. This inhibition is mediated by direct binding of merlin to DDB1-and-Cullin 4-associated Factor 1 (DCAF1), yet the binding mode of merlin to DCAF1 is not well defined. Here, we report structural and biophysical studies of the merlin binding to DCAF1 and its interference with CD44 binding. The crystal structure of the merlin FERM domain bound to the DCAF1 C-terminal acidic tail reveals that the hydrophobic IILXLN motif located at the C-terminal end of DCAF1 binds subdomain C of the FERM domain by forming a β-strand. The binding site and mode resemble that of merlin binding to the CD44 cytoplasmic tail. Competition binding assay showed that CD44 and DCAF1 compete for binding to the merlin FERM domain in solution. The CD44 cytoplasmic tail is known to be cleaved for nuclear translocation by regulated intra-membrane proteolysis (RIP). Our structure implies that, in the nucleus, the CD44 cytoplasmic tail cleaved by RIP could release DCAF1 from merlin by competing for binding to the merlin FERM domain, which results in the inhibition of merlin-mediated suppression of tumorigenesis.