Functional interaction of CD154 protein with α5β1 integrin is totally independent from its binding to αIIbβ3 integrin and CD40 molecules

In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ～4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.     

Overexpression of cellular prion protein induces an antioxidant environment altering T cell development in the thymus

Cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein whose roles are still widely discussed, particularly in the field of immunology. Using TgA20- and Tg33-transgenic mice overexpressing PrP(C), we investigated the consequences of this overexpression on T cell development. In both models, overexpression of PrP(C) induces strong alterations at different steps of T cell maturation. On TgA20 mice, we observed that these alterations are cell autonomous and lead to a decrease of alphabeta T cells and a concomitant increase of gammadelta T cell numbers. PrP(C) has been shown to bind and chelate copper and, interestingly, under a copper supplementation diet, TgA20 mice presented a partial restoration of the alphabeta T cell development, suggesting that PrP(C) overexpression, by chelating copper, generates an antioxidant context differentially impacting on alphabeta and gammadelta T cell lineage.     

Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone

The ClpXP ATPase-protease complex is a major component of the protein quality control machinery in the cell. A ClpX subunit consists of an N-terminal zinc binding domain (ZBD) and a C-terminal AAA+ domain. ClpX oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the ZBDs extending out of the base. Here, we report that ClpX switches between a capture and a feeding conformation. ZBDs in ClpX undergo large nucleotide-dependent block movement towards ClpP and into the AAA+ ring. This motion is modulated by the ClpX cofactor, SspB. Evidence for this movement was initially obtained by the surprising observation that an N-terminal extension on ClpX is clipped by bound ClpP in functional ClpXP complexes. Protease-protection, crosslinking, and light scattering experiments further support these findings.     

Antigen presenting cells (APCs) from thermally injured and/or septic rats modulate CD4+ T cell responses of naive rat

Regulation of immune response is marked by complex interactions among the cells that recognize and present antigens. Antigen presenting cells (APCs), the antigen presenting cell component of the innate immune response plays an important role in effector CD4+ T cell response. Thermal injury and/or superimposed sepsis in rats' leads to suppressed CD4+ T cell functions. We investigated modulations of CD4+ T cell function by APCs (purified non-T cells) from thermally injured and/or septic rats. Rats were subjected to 30% total body surface area scald burn or exposed to 37 degrees C water (Sham burn) and sepsis was induced by cecal-ligation and puncture (CLP) method. At day 3 post-injury animals were sacrificed and CD4+ T cells and APCs from mesenteric lymph nodes (MLN) were obtained using magnetic microbead isolation procedure. APCs from injured rats were co-cultured with sham rat MLN CD4+ T cells and proliferative responses (thymidine incorporation), phenotypic changes (Flow cytometry), IL-2 production (ELISA) and CTLA-4 mRNA (RT-PCR) were determined in naive rat CD4+ T cells. The data indicate that APCs from thermally injured and/or septic rats when co-cultured with CD4+ T cells suppressed CD4+ T cell effector functions. This lack of CD4+ T cell activation was accompanied with altered co-stimulatory molecules, i.e., CD28 and/or CTLA-4 (CD152). In conclusion, our studies indicated that defective APCs from thermally injured and/or septic rats modulate CD4+ T cell functions via changes in co-stimulatory molecules expressed on naive CD4+ T cells. This altered APC: CD4+ T cell interaction leads to suppressed CD4+ T cell activation of healthy animals.     

Agrin Regulation of ��3 Sodium-Potassium ATPase Activity Modulates Cardiac Myocyte Contraction

Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of α3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the α3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the α3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na⁺ in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-α3 Na,K-ATPase interaction and suggest that agrin modulation of the α3 Na,K-ATPase is important in regulating heart function.Na,K-ATPases, or sodium pumps, are integral membrane enzymes found in all animal cells. Using energy from the hydrolysis of ATP they transport three Na⁺ ions out of the cell for every two K⁺ ions into the cell, resulting in a transmembrane chemical gradient that is reflected in the resting membrane potential and used to drive a variety of secondary transport processes. Each Na,K-ATPase is a heterodimer consisting of an α- and β-subunit. The α-subunit is the catalytic subunit and contains the binding sites for Na⁺ and K⁺. The β-subunit is required for pump function and targeting of the α-subunit to the plasma membrane. Four α- and three β-subunit genes have been identified. All combinations of α- and β-subunits form functional pumps, but developmental, cellular, and subcellular differences in expression suggest functional adaptation of the different isoforms (1).Na,K-ATPases play a central role in regulating the contractile activity of cardiac muscle (2). They are directly responsible for the Na⁺ gradient required for propagation of action potentials that initiate myocyte contraction. Moreover, because of the dependence of the Na⁺/Ca²⁺ exchanger (NCX)³ on the Na⁺ gradient as the source of counterions for transport of Ca²⁺ out of the cell, they play a critical role in Ca²⁺ homeostasis and excitation-contraction coupling. For example, inhibition of Na,K-ATPases by digoxin, ouabain, or other cardiac glycoside results in a decline of the Na⁺ gradient, reducing NCX activity and Ca²⁺ efflux. The inotropic effects of cardiac glycosides result from uptake of this “excess” cytoplasmic Ca²⁺ into the sarcoplasmic reticulum, raising the level of Ca²⁺ in intracellular stores, which, when released during excitation, enhances muscle contraction (3).In light of the importance of Na,K-ATPases for cardiac muscle function, it is not surprising that mechanisms have evolved to regulate their activity. Na,K-ATPases are susceptible to phosphorylation by either cAMP-dependent protein kinase or protein kinase C, and neurotransmitter- and peptide hormone-dependent activation of these cytoplasmic kinases have been shown to regulate pump activity (4). Other molecules exert their effects through direct interaction with the Na,K-ATPase. For example, phospholemman, a member of the FXYD family of membrane proteins expressed in heart, is tightly associated with the Na,K-ATPase and inhibits its function (5–7). Phosphorylation of phospholemman by either protein kinase C or cAMP-dependent protein kinase, however, relieves inhibition thereby restoring the activity of the pump (8, 9). Endogenous ouabain-like compounds have also been implicated in regulating Na,K-ATPase activity (10). Ouabain, or closely related molecules, is synthesized by the adrenal gland and hypothalamus, and increased circulating levels of these compounds observed in patients with congestive heart failure has been suggested as an adaptive response to improve heart function (11). Recent studies in the central nervous system have identified the protein agrin as a new endogenous ligand that regulates Na,K-ATPase function through interaction with its extracellular domains (12).Agrin was first identified as an extracellular matrix protein at the neuromuscular junction where, by signaling through a muscle-specific receptor tyrosine kinase called MuSK, it mediates the motor neuron-induced accumulation of acetylcholine receptors in the postsynaptic muscle fiber membrane (13). Agrin is also expressed in other tissues (14–16), but its function outside of the neuromuscular junction has been less well understood. Recently, however, we showed that agrin plays a role in regulating excitability of central nervous system neurons by binding to and inhibiting the activity of the α3 subunit-containing isoform of the Na,K-ATPase (12). Although both agrin (14, 16) and the α3 Na,K-ATPase (17) are expressed in heart, their potential interaction has not been explored. Here we show that the frequency of cardiac myocyte contraction is modulated by agrin regulation of α3 Na,K-ATPase activity.     

Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4

Background

The human endogenous retrovirus HERV-K(HML-2) family is associated with testicular germ cell tumors (GCT). Various HML-2 proviruses encode viral proteins such as Env and Rec.

Results

We describe here that HML-2 Env gives rise to a 13 kDa signal peptide (SP) that harbors a different C-terminus compared to Rec. Subsequent to guiding Env to the endoplasmatic reticulum (ER), HML-2 SP is released into the cytosol. Biochemical analysis and confocal microscopy demonstrated that similar to Rec, SP efficiently translocates to the granular component of nucleoli. Unlike Rec, SP does not shuttle between nucleus and cytoplasm. SP is less stable than Rec as it is subjected to proteasomal degradation. Moreover, SP lacks export activity towards HML-2 genomic RNA, the main function of Rec in the original viral context, and SP does not interfere with Rec's RNA export activity.

Conclusion

SP is a previously unrecognized HML-2 protein that, besides targeting and translocation of Env into the ER lumen, may exert biological functions distinct from Rec. HML-2 SP represents another functional similarity with the closely related Mouse Mammary Tumor Virus that encodes an Env-derived SP named p14. Our findings furthermore support the emerging concept of bioactive SPs as a conserved retroviral strategy to modulate their host cell environment, evidenced here by a "retroviral fossil". While the specific role of HML-2 SP remains to be elucidated in the context of human biology, we speculate that it may be involved in immune evasion of GCT cells or tumorigenesis.