Selective activation of distinct Orai channels by STIM1

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Selective activation by thymus-dependent antigens of distinct B cell subpopulations expressing a major cross-reactive idiotype

We determined the B cell subpopulations that produce the major cross-reactive idiotype (CRIA) associated with the anti-phenylarsonate (ARS) antibody response of A/J mice. Specifically, we examined the B2 subpopulation found in normal mice which, in H-2b mice, bears the I-Ab-encoded determinant Ia.W39; the B1 subpopulation found in mice expressing the CBA/N X-linked immunodeficiency trait (xid); and the B1 subpopulation found in normal mice after the cytotoxic elimination of B2 cells with anti-Ia. W39 and complement. CRIA is expressed in each of these B cell subpopulations. Antigen plays a selective role in the stimulation of distinct B cell sets. ARS conjugates of keyhole limpet hemocyanin (KLH) can activate both the B1 and B2 subpopulations. In contrast, ARS conjugates of synthetic polypeptides under Ir gene control selectively activate the B2 subpopulation in strains that are genetic responders to the carrier. This leads to the establishment of CRIA dominance where CRIA+ anti-ARS antibody is 70 to 95% of the total anti-arsonate antibody response. This class of antigens fails to activate the B1 cells in either normal or xid mice. We compared the CRIA+ antibody produced by selectively activated B2 cells to that produced by the B1 subpopulation in xid mice. For these comparisons, we used competitive radioimmunoassays that employed polyspecific anti-CRIA antiserum or monoclonal anti-CRIA antibodies specific for distinct idiotopes on the heavy chain of CRIA+ antibody. B2 cells produce a CRIA+ anti-ARS antibody that is idiotopically uniform among individual mice, and that closely approximates the hybridoma protein 36-65 (the heavy chain of 36-65 represents the germ line-encoded sequence of the unique CRIA structural gene (25]. In contrast, the CRIA+ antibody produced by the B1 cell subset of xid mice is idiotopically diverse among individual mice, and differs markedly from the 36-65 hybridoma protein. The extent of diversification found in CRIA+ antibody depends on the B cell subpopulation that produces it.     

Signaling by distinct classes of phosphoinositide 3-kinases

Many signaling pathways converge on and regulate phosphoinositide 3-kinase (PI3K) enzymes whose inositol lipid products are key mediators of intracellular signaling. Different PI3K isoforms generate specific lipids that bind to FYVE and pleckstrin homology (PH) domains in a variety of proteins, affecting their localization, conformation, and activities. Here we review the activation mechanisms of the different types of PI3Ks and their downstream actions, with focus on the PI3Ks that are acutely triggered by extracellular stimulation.     

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem-iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem-binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter-like beta-sandwich fold with a large hydrophobic haem-binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co-ordinated solely by Tyr(166). The phenol groups of Tyr(166) and Tyr(170) form an H-bond that may function in regulating haem binding and release. An analysis of IsdA structure-sequence alignments indicate that conservation of Tyr(166) is a predictor of haem binding by NEAT domains.     

RNA recognition by a Staufen double-stranded RNA-binding domain

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix α1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.     

The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation

A 60 amino acid domain of the myogenic determination gene MyoD is necessary and sufficient for sequence-specific DNA binding in vitro and myogenic conversion of transfected C3H10T1/2 cells. We show that a highly basic region, immediately upstream of the helix-loop-helix (HLH) oligomerization motif, is required for MyoD DNA binding in vitro. Replacing helix1, helix2, or the loop of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (required for peripheral neurogenesis) has no substantial effect on DNA binding in vitro or muscle-specific gene activation in transfected C3H10T1/2 cells. However, replacing the basic region of MyoD with the analogous sequence of other HLH proteins (the immunoglobulin enhancer binding E12 protein or T4 achaete scute protein) allows DNA binding in vitro, yet abolishes muscle-specific gene activation. These findings suggest that a recognition code that determines muscle-specific gene activation lies within the MyoD basic region and that the capacity for specific DNA binding is insufficient to activate the muscle program.     

Selective ligand recognition by a diversity-generating retroelement variable protein

Diversity-generating retroelements (DGRs) recognize novel ligands through massive protein sequence variation, a property shared uniquely with the adaptive immune response. Little is known about how recognition is achieved by DGR variable proteins. Here, we present the structure of the Bordetella bacteriophage DGR variable protein major tropism determinant (Mtd) bound to the receptor pertactin, revealing remarkable adaptability in the static binding sites of Mtd. Despite large dissimilarities in ligand binding mode, principles underlying selective recognition were strikingly conserved between Mtd and immunoreceptors. Central to this was the differential amplification of binding strengths by avidity (i.e., multivalency), which not only relaxed the demand for optimal complementarity between Mtd and pertactin but also enhanced distinctions among binding events to provide selectivity. A quantitatively similar balance between complementarity and avidity was observed for Bordetella bacteriophage DGR as occurs in the immune system, suggesting that variable repertoires operate under a narrow set of conditions to recognize novel ligands.     

The transcriptional coactivators SAGA, SWI/SNF, and mediator make distinct contributions to activation of glucose-repressed genes

The paradigm of activation via ordered recruitment has evolved into a complicated picture as the influence of coactivators and chromatin structures on gene regulation becomes understood. We present here a comprehensive study of many elements of activation of ADH2 and FBP1, two glucose-regulated genes. We identify SWI/SNF as the major chromatin-remodeling complex at these genes, whereas SAGA (Spt-Ada-Gcn5-acetyltransferase complex) is required for stable recruitment of other coactivators. Mediator plays a crucial role in expression of both genes but does not affect chromatin remodeling. We found that Adr1 bound unaided by coactivators to ADH2, but Cat8 binding depended on coactivators at FBP1. Taken together, our results suggest that commonly regulated genes share many aspects of activation, but that gene-specific regulators or elements of promoter architecture may account for small differences in the mechanism of activation. Finally, we found that activator overexpression can compensate for the loss of SWI/SNF but not for the loss of SAGA.