Signaling Signatures and Functional Properties of Anti-Human CD28 Superagonistic Antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4+ T cells but not in cynomolgus T lymphocytes. The sustained Ca++-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells

HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion

BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/-) mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer

Functional characterization of the human myosin-7a motor domain

Myosin-7a participates in auditory and visual processes. Defects in MYO7A, the gene encoding the myosin-7a heavy chain, are causative for Usher syndrome 1B, the most frequent cause of deaf-blindness in humans. In the present study, we performed a detailed kinetic and functional characterization of the isolated human myosin-7a motor domain to elucidate the details of chemomechanical coupling and the regulation of motor function. A rate-limiting, slow ADP release step causes long lifetimes of strong actin-binding intermediates and results in a high duty ratio. Moreover, our results reveal a Mg2+-sensitive regulatory mechanism tuning the kinetic and mechanical properties of the myosin-7a motor domain. We obtained direct evidence that changes in the concentration of free Mg2+ ions affect the motor properties of human myosin-7a using an in vitro motility assay system. Our results suggest that in a cellular environment, compartment-specific fluctuations in free Mg2+ ions can mediate the conditional switching of myosin-7a between cargo moving and tension bearing modes

Stepwise modulation of ATPase activity, nucleotide trapping, and sliding motility of myosin S1 by modification of the thiol region with residues of increasing size.

Rabbit muscle myosin S1 was modified either at SH1 alone or at both SH1 and SH2, using a series of alkylthiolating reagents of increasing size, designed for correlating gradually changing structural disturbances in the thiol region with functional impairments in the myosin head. The reagents were of the type H(CH(2))(n)()-S-NTB, (NTB = 2-nitro-5-thiobenzoate) (n = 1, 2, 5, 8, 9, 10, 11, and 12). Modification of only SH1 led to the expected activation of the Ca(2+)-ATPase, but only with small reagents, while reagents with n > or = 10 caused inhibition of the Ca(2+)-ATPase. Modification of both SH1 and SH2 showed the expected inhibition of Ca(2+)-ATPase but likewise allowed considerable residual Ca(2+)-ATPase activity if the residues were small. Trapping of the nucleotide, known to occur with cross-linking reagents, was seen also with monovalent reagents, provided their length exceeded n = 9 or 10. All S1 derivatives prepared in this study possessed an affinity for actin comparable to native S1 but lacked sliding motility in in vitro motility assays. The biochemical data of this study can be related to existing models of myosin S1 and recent structural data [Houdusse, A., Kalabokis, V. N., Himmel, D., Szent-Györgyi, A. G., and Cohen, C. (1999) Cell 97, 459-470] by making the assumptions that modification at SH1 prevents the formation of the SH1 helix mandatory for the transmission of conformational energy and that mobility of the thiol region is a prerequisite for ATPase activity. Immobilization of the thiol region by residues of increasing size apparently leads to lower enzyme activity and, finally, to inhibition of nucleotide exchange

Thiol-specific cross-linkers of variable length reveal a similar separation of SH1 and SH2 in myosin subfragment 1 in the presence and absence of MgADP

A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys707) and SH2 (Cys697) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH2)nSSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 ?. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k1) and for cross-linking (k2) were measured by the decrease of the K+(EDTA)-ATPase activity and the decrease of the Ca2+-ATPase activity, respectively, and corrected for the different reactivities of Cn. Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellmans reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as 15 ?, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k2 for C3, C6, and C7 in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628

Crystallization and preliminary X-ray analysis of UMP/CMP-kinase from Dictyostelium discoideum with the specific bisubstrate inhibitor P1-(adenosine 5′)-P5-(uridine 5′)-pentaphosphate (UP5A)

UMP/ICMP-kinase (UK) from the slime mold Dictyostelium discoideum has been purified to high homogeneity and co-crystallized with the bisubstrate inhibitor P1-(adenosine 5′)-P5-(uridine 5′)-pentaphosphate (UP5A). UP5A binds to UK with a dissociation constant (K d ) of 3 ± 0.5 nM at 25°C and pH 7.5. This is some 50-fold tighter than the binding of P1,P5-(diadenosine 5′)-pentaphosphate (AP5A, K d = 160 ± 15 nM). AP5A is a bisubstrate inhibitor that is specific for adenylate kinase. The crystals have the symmetry of the tetragonal space group P41212 or its enantiomorph P43212. The unit cell dimensions are math formula. The crystals diffract to a Bragg spacing of 2.1 Å