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In B lymphocytes, Ig class switch recombination (CSR) is induced by activation-induced cytidine deaminase, which initiates a cascade of events leading to DNA double-strand break formation in switch (S) regions. Resolution of DNA double-strand breaks proceeds through formation of S-S synaptic complexes. S-S synapsis is mediated by a chromatin loop that spans the C region domain of the Igh locus. S-S junctions are joined via a nonhomologous end joining DNA repair process. CSR occurs via an intrachromosomal looping out and deletion mechanism that is 53BP1 dependent. However, the mechanism by which 53BP1 facilitates deletional CSR and inhibits inversional switching events remains unknown. We report a novel architectural role for 53BP1 in Igh chromatin looping in mouse B cells. Long-range interactions between the Eμ and 3'Eα enhancers are significantly diminished in the absence of 53BP1. In contrast, germline transcript promoter:3'Eα looping interactions are unaffected by 53BP1 deficiency. Furthermore, 53BP1 chromatin occupancy at sites in the Igh locus is B cell specific, is correlated with histone H4 lysine 20 marks, and is subject to chromatin spreading. Thus, 53BP1 is required for three-dimensional organization of the Igh locus and provides a plausible explanation for the link with 53BP1 enforcement of deletional CSR., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Ca2+-binding protein-1 (CaBP1) is a Ca2+-binding protein that is closely related to calmodulin (CaM) and localized in somatodendritic regions of principal neurons throughout the brain, but how CaBP1 participates in postsynaptic Ca2+ signaling is not known. Here, we describe a novel role for CaBP1 in the regulation of Ca2+ influx through Ca(v)1.2 (L-type) Ca2+ channels. CaBP1 interacts directly with the alpha1 subunit of Ca(v)1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Ca(v)1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Ca(v)1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Ca(v)1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+-dependent inactivation of Ca(v)1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Ca(v)1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons.

This study analyses by immunohistochemical methods the effects of the deletion of the Otx1 gene on 12 areas of the cerebral cortex and on neurons expressing Ca-binding proteins (CaBP), such as parvalbumin (Pv) and calbindin-D28K (Cb). We found that the deletion of the Otx1 gene modified differently the various cortical areas. The decrease in cortical thickness ranged from 29.35 to 9.85% and the reduction in cellular population from 35.90 to 3.65% in the different cortical areas. The influence of the Otx1 gene concerns all cortical layers with variable effects on different cortical areas. The cellular population of cerebral cortex considered as a whole was reduced by 20.67%, Pv-positive (Pv+) cells by 58.01% and Cb-positive (Cb+) cells by 51.54%. The quantitative distribution of Pv+ and Cb+ cells varied independently in the different cortical areas. Topographic analysis of CaBP cells in Otx1-null mice (Otx1(-/-)) showed that Pv+ cells were principally distributed in layers IV and V and Cb+ cells in layers V and VI. Given that in the development of wild-type mice both cell types first appear in deep layers and later spread to superficial ones, the segregation of CaBP neurons in inner layers of Otx1(-/-) animals is an index of the immaturity of the cerebral cortex of these animals. This study showed that the Otx1 gene has a more complex role than previously reported, as it is involved in the maturation and differentiation of various cerebral cortices, and, specifically, in the development of CaBP cells.

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.

In recent years, along with the rapid development of genetic engineering technologies, a vast amount of valuable proteins have been expressed in E. coli with high productivity. The problems concerning the productivity and purity of the heterologous proteins are no longer the major ones, as many expression system and protein purification schemes have been developed and perfected. More attention is being payed to the activity, specific activity, correct folding and intactness of the heterologous proteins. Perhaps it is the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins.

The ER retention of lumenal proteins is achieved by a process which involves binding of escaped proteins via the C-terminal KDEL-tags to a KDEL receptor (erd2 receptor) in a post-ER compartment and return of the protein-receptor complex back to the ER. The transmembrane topology of the human KDEL receptor, which is an integral membrane protein, has been proposed. We have synthesised sets of cellulose-bound overlapping peptides covering the complete se quence of the receptor to study the interaction of the erd2 receptor with lumenal ER proteins, CaBP1 and CaBP2. At the next stage, the proposed lumenal loops of the receptor were more closely mapped. A short sequence, essential for the protein binding to the most efficient binding site of the receptor, was identified as 22KIWK25, which is in accordance with one of the proposed structural models of the receptor. The binding was of high specificity and was almost completely inhibited by KDEL-containing soluble peptides. The phosphorylation state of CaBP1/CaBP2 did not affect their binding to the KDEL receptor., (Copyright 1998 Academic Press.)

Background: Disulfide exchange reactions are catalyzed by thiol/disulfide oxidoreductases. These enzymes possess a thioredoxin fold and contain a catalytic disulfide with the sequence Cys-X-X-Cys at the N terminus of an alpha helix. Despite these similarities, the various members differ strongly in their redox potentials (-122 mV to -270 mV). Using the strong oxidant DsbA from Escherichia coli as a model system, we investigated whether the redox properties of these enzymes can be modulated rationally by exchange of the X-X dipeptide., Results: The X-X dipeptide of DsbA (Cys30-Pro31-His32-Cys33) was exchanged by the dipeptides of eukaryotic protein disulfide isomerase (PDI; Gly-His), glutaredoxin (Pro-Tyr), and thioredoxin (Gly-Pro) from E. coli. All variants were less oxidizing than wild-type DsbA and their redox potentials were in the order of the related natural enzymes (DsbA > PDI > glutaredoxin > thioredoxin). The equilibrium constant between glutathione and the thioredoxin-like variant increased 1200-fold compared with wild-type DsbA. The variants also showed a strong increase in the pKa of the nucleophilic cysteine (Cys30). As for glutaredoxin and thioredoxin, the catalytic disulfide stabilized the corresponding variants while destabilizing wild-type DsbA and the PDI-like variant., Conclusions: The X-X dipeptide in the active site of thiol/disulfide oxidoreductases appears to be the main determinant of the redox properties of these enzymes. This empirical finding should be very useful for the design of new thiol/disulfide oxidoreductases with altered redox potentials and for studying the function of these enzymes in vivo.