Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor binding site of the protein

Inducible costimulator (ICOS) ligand (ICOSL), a B7-related transmembrane glycoprotein with extracellular IgV and IgC domains, binds to ICOS on activated T cells and delivers a positive costimulatory signal for optimal T cell function. Toward determining the structural features of ICOSL crucial for its costimulatory function, the present study shows that ICOSL displays a marked oligomerization potential, resembling more like B7-1 than B7-2. Use of ICOSL constructs lacking either the IgC or IgV domain demonstrates that receptor binding is mediated solely by the IgV domain but requires the IgC domain for maintaining the structural integrity of the protein. To map further the receptor recognition surface on ICOSL, a homology-based protein structure model of the ICOS:ICOSL complex was constructed. Based on predictions from the model, a series of mutations were generated targeting the potential receptor binding surface on ICOSL, and the mutants were tested for their biological function in terms of ICOS binding and T cell costimulation ability. The results provide experimental validation of the model and show that the receptor binding site on ICOSL is constituted chiefly by aromatic/hydrophobic residues. Critical ICOSL residues essential for ICOS binding map to the GFCC'C' beta-sheet face of the IgV domain and approximately overlap with the B7-1/B7-2 motif(s) that recognize CD28/CTLA-4. Altogether, similar structural features of ICOSL and B7 isoforms suggest a close evolutionary relationship between these costimulatory ligands, yet differences at the same time explain their unique specificity for the cognate binding partners, ICOS and CD28/CTLA-4, respectively.     

Cooperative effects of cofilin (ADF) on actin structure suggest allosteric mechanism of cofilin function

Using site-specific fluorescence probes and cross-linking we demonstrated that cofilin (ADF), a key regulator of actin cellular dynamics, weakens longitudinal contacts in F-actin in a cooperative manner. Differential scanning calorimetry detected a dual nature of cofilin effects on F-actin conformation. At sub-stoichiometric cofilin to actin ratios, cofilin stabilized sterically and non-cooperatively protomers at the points of attachment, and destabilized allosterically and cooperatively protomers in the cofilin-free parts of F-actin. This destabilizing effect had a long range, with one cofilin molecule affecting more than 100 protomers, and concentration-dependent amplitude that reached maximum at about 1:2 molar ratio of cofilin to actin. In contrast to existing models, our results suggest an allosteric mechanism of actin depolymerization by cofilin. We propose that cofilin is less likely to sever actin filaments at the points of attachment as thought previously. Instead, due to its dual structural effect, spontaneous fragmentation occurs most likely in cofilin-free segments of filaments weakened allosterically by nearby cofilin molecules.     

Nitric oxide, mitochondrial hyperpolarization, and T cell activation

T lymphocyte activation is associated with nitric oxide (NO) production, which plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both activation and apoptosis of Tlymphocytes, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus induces mitochondrial biogenesis and alters Ca(2+) signaling. Thus, whereas NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity.     

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 ?) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.     

Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking

Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1(-/-)). HIP1(-/-) mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1(-/-) mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis.     

The pleckstrin homology domain of phosphoinositide-specific phospholipase Cdelta4 is not a critical determinant of the membrane localization of the enzyme

The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.