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Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker™ Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.Cell Death and Differentiation (2007) 14, 733–742. doi:10.1038/sj.cdd.4402066; published online 15 December 2006 [ABSTRACT FROM AUTHOR]

The adaptor protein Swiprosin-1/Efhd2 that we identified recently in B cell lipid rafts exhibits its highest expression in immature B cells. Swiprosin-1/Efhd2 consists of a proline rich domain, two EF-hands and a C-terminal coiledcoil domain. Knock-down of Swiprosin-1/Efhd2 in an immature B cell line with a shRNA directed against the 3'-UTR (WEHI231shSw1) impaired spontaneous and BCR-elicited apoptosis. Due to co-clustering of a Swiprosin-1-EGFP fusion protein with the B-cell receptor we expected Swiprosin-1 to be a player in proximal BCR signaling. Indeed, downregulation of Swiprosin-1 attenuated total tyrosine phosphorylation and diminished BCR induced intracellular calcium flux. In contrast, WEHI231 cells ectopically expressing Swiprosin-1 showed increased, but shortened total tyrosine phosphorylation at early time points, as well as increased BCR induced calcium flux. Concomitantly, transient re-expression of Swiprosin-1 in WEHI231shSW1 cells restored BCR-induced calcium flux. These data suggested interactions of Swiprosin-1 with central elements of the BCR signaling cascade. In fact, GST-pulldown experiments showed that Swiprosin-1 can interact with tyrosine-phosphorylated proteins of ~145 and 70 kDa. With this assay we identified the protein tyrosine kinase Syk in its phosphorylated form as interaction partner of Swiprosin-1. The interaction of pSyk with Swiprosin-1 is mediated by the C-terminal part of Swiprosin-1 because a deletion mutant of Swiprosin-1 lacking the EF-hands and the coiled-coil domain of Swiprosin-1 did not show any interaction with pSyk. We next tested the kinase activity of Syk as a function of Swiprosin-1 levels. Interestingly, immune complex kinase assays showed that BCR-induced Syk activity was reduced in WEHI231shSw1 cells Hence, Swiprosin-1 could regulate proximal BCR signaling through maintenance of Syk activity. [ABSTRACT FROM AUTHOR]

Carma-1 is required for B cell receptor-/CD40- and T cell receptor-/CD28-induced B- and T-cell activation via JNK and NF-βB. In B cells, Carma-1 becomes phosphorylated by PKCβ, leading to its oligomerization. Subsequent Bcl10 binding induces IKKβ-activation and, thereby, canonical NF-κB signalling. Despite these findings it is still unknown how exactly Carma-1 is connected to the plasma membrane and to the IKK-complex. Therefore, we purified Carma-1 complexes from mouse CH12 B cells using anti-Carma-1 affinity columns. Mass spectrometric analyses of the column eluates demonstrated the presence of Carma-1 as well as three previously uncharacterized adaptor proteins in B cells, one of which was the Trk-fused gene (Tfg), an adaptor protein containing PB1 and coiledcoil domains. Whereas Tfg was originally identified as fusion partner of oncogenic Trk tyrosine kinase mutants, the normal cellular homologue of Tfg has so far not been described in B cells. However, Tfg has been shown in other systems to interact with IKKγ and to enhance TNF-induced NF-κB activation. Tfg and Carma-1 co-localized at the plasma membrane and perinuclear structures in B cells. We further corroborated the interactions of Tfg, IKKγ and Carma-1 by Blue Native gel electrophoresis, where Carma-1 and Tfg formed a 0.7-1 MDa complex. Ectopic expression of Tfg increased the molecular mass of IKKγ complexes, fused IKKγ, Bcl10 and Carma-1 complexes to a ~2 MDa complex, and increased basal and CD40-induced canonical activity of NF-κB and IKKβ. In contrast, shRNA-mediated silencing of Tfg decreased CD40-induced IKKβ activity. Very interestingly, in primary B cells, highest expression of Tfg was detected in marginal zone and B1 B cells, and Carma-1 and Tfg formed complexes in these B cells. Since Carma-1 is required for marginal zone B cell and B1 B cell development, we suggest that a functional interaction between Carma-1 and Tfg contributes to development and maintenance of these cells by means of canonical NF-κB signals. [ABSTRACT FROM AUTHOR]

A correction to the article "After shrinkage apoptotic cells expose internal membrane-derived epitopes on their plasma membranes," is presented.

The potency of antibody neutralization in cell culture has been used as the key criterion for selection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for clinical development. As other aspects may also influence the degree of protection in vivo , we compared the efficacy of two neutralizing monoclonal antibodies (TRES6 and 4C12) targeting different epitopes of the receptor binding domain (RBD) of SARS-CoV-2 in a prophylactic setting in rhesus monkeys. All four animals treated with TRES6 had reduced viral loads in the upper respiratory tract 2 days after naso-oropharyngeal challenge with the Alpha SARS-CoV-2 variant. Starting 2 days after challenge, mutations conferring resistance to TRES6 were dominant in two of the rhesus monkeys, with both animals failing to maintain reduced viral loads. Consistent with its lower serum neutralization titer at the day of challenge, prophylaxis with 4C12 tended to suppress viral load at day 2 less efficiently than TRES6. However, a week after challenge, mean viral loads in the lower respiratory tract in 4C12-treated animals were lower than in the TRES6 group and no mutations conferring resistance to 4C12 could be detected in viral isolates from nasal or throat swabs. Thus, genetic barrier to resistance seems to be a critical parameter for the efficacy of prophylaxis with monoclonal antibodies against SARS-CoV-2. Furthermore, comparison of antibody concentrations in respiratory secretions to those in serum shows reduced distribution of the 4C12 antibody into respiratory secretions and a delay in the appearance of antibodies in bronchoalveolar lavage fluid compared to their appearance in secretions of the upper respiratory tract.IMPORTANCEMonoclonal antibodies are a powerful tool for the prophylaxis and treatment of acute viral infections. Hence, they were one of the first therapeutic agents licensed for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oftentimes, the main criterion for the selection of antibodies for clinical development is their potency of neutralization in cell culture. By comparing two antibodies targeting the Spike protein of SARS-CoV-2, we now observed that the antibody that neutralized SARS-CoV-2 more efficiently in cell culture suppressed viral load in challenged rhesus monkeys to a lesser extent. Extraordinary rapid emergence of mutants of the challenge virus, which had lost their sensitivity to the antibody, was identified as the major reason for the reduced efficacy of the antibody in rhesus monkeys. Therefore, the viral genetic barrier to resistance to antibodies also affects their efficacy., Competing Interests: The authors declare no conflict of interest.

The assembly of a pre-B cell receptor (pre-BCR) composed of an Ig mu heavy chain (mu H-chain), the surrogate light (SL) chain, and the Ig alpha/beta dimer is critical for late pro-B cells to advance to the pre-B cell stage. By using a transgenic mouse model, in which mu H-chain synthesis is solely driven by a tetracycline-controlled transactivator, we show that de novo synthesis of mu H-chain in transgenic pro-B cells not only induces differentiation but also proliferation. This positive effect of mu H-chain synthesis on proliferation requires the presence of SL chain and costimulatory signals provided by stromal cells or IL-7. We conclude that pre-BCR signaling induces clonal expansion of early pre-B cells.

CD30 engagement in human lymphoid cells induces pleiotropic cellular responses that affect cellular viability and proliferation, cytokine production, and nuclear factor kappaB (NF-kappaB) nuclear translocation. Studies examining the molecular basis for this pleiotropism thus far have relied on the use of antibodies and cells transfected with CD30L to trigger CD30, two methods of receptor induction that present important limitations: antibodies are not physiological receptor-triggering molecules and CD30L transfectants induce high background intracellular signaling in the cells under study. We have generated and expressed a functional soluble human CD30L molecule (sCD30L/CD8alpha) comprised of the extracellular domain of human CD30L fused to the extracellular domain of the human CD8alpha chain. Immunoprecipitation and Western blot analysis of sCD30L/CD8alpha revealed the existence of at least two forms of sCD30L/CD8alpha, which exhibited molecular sizes consistent with the existence of monomeric and trimeric forms of the molecule. Binding analyses performed using a soluble CD30 fusion protein (sCD30/gamma1) confirmed the ability of sCD30L/CD8alpha to bind to CD30. Functionally, immobilized sCD30L/CD8alpha-induced cell death in the CD30-expressing lines Karpas-299 and HDLM-2 and reduced proliferative levels in Karpas-299; these effects were inhibitable by the addition of sCD30/gamma1. These studies demonstrate the utility of sCD30L/CD8alpha in characterizing the normal function of CD30L and CD30 and indicate the natural ability of soluble forms of CD30L to trimerize.

Levels of most nonsense mRNAs are normally reduced in prokaryotes and eukaryotes when compared with that of corresponding functional mRNAs. Genes encoding polypeptides that selectively reduce levels of nonsense mRNA have so far only been identified in simple eukaryotes. We have now cloned a human cDNA whose deduced amino acid sequence shows the highest degree of homology to that of UPF1, a bona fide Saccharomyces cerevisiae group I RNA helicase required for accelerated degradation of nonsense mRNA. Based on the total sequence of the shorter yeast UPF1 protein, the overall identity between the human protein and UPF1 is 51%. Besides NTPase and other RNA helicase consensus motifs, UPF1 and its human homolog also share similar putative zinc finger motifs that are absent in other group I RNA helicases. Northern blot analysis with the human cDNA probe revealed two transcripts in several human cell lines. Further, antibodies raised against a synthetic peptide of the human polypeptide detected a single 130 kDa polypeptide on Western blots from human and mouse cells. Finally, immunofluorescence and Western blot analyses revealed that the human and mouse polypeptides, like yeast UPF1, are expressed in the cytoplasm, but not in the nucleus. We have thus identified the first mammalian homolog of yeast UPF1, a protein that regulates levels of nonsense mRNA, and we tentatively name this protein human HUPF1 (for human homolog of UPF1).

Background: The immunoglobulin framework has been mutagenized to engineer recombinant libraries of proteins as potential diagnostics and novel catalysts, although the often shallow binding cleft may limit the utility of this framework for binding diverse small organic molecules. By contrast, the glutathione S-transferase (GST) family of enzymes contains a deep binding cleft, which has evolved to accommodate a broad range of hydrophobic xenobiotics. We set out to determine whether GST molecules with novel ligand-binding characteristics could be produced by random mutagenesis of segments of the binding cleft., Results: We have identified two ligand-recognition segments (LRSs) in human GST P1, which are near the active site in the folded protein, but have characteristics indicating that the integrity of their sequence is not essential for the overall structure or activity of the protein. Libraries of GST P1-derived proteins were produced by substituting randomized sequences for an LRS or inserting random sequences into an LRS. The recombinant proteins in the libraries, collectively designated as 'glubodies,' generally retain enzymatic activity but differ markedly both from each other and from the parent enzyme in sensitivity to inhibition by diverse small organic compounds. In some instances, a glubody is inhibited by completely novel structures., Conclusions: We have shown that a non-antibody framework can be used to create large libraries of proteins with a wide range of binding specificities for small organic molecules. The glubodies provide a rich source of data for correlating the structural and functional features of proteins relevant to ligand binding. The criteria applied for identifying an LRS in GST P1 are generally applicable to other protein frameworks.

Two full-length cDNA clones encoding rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were isolated from a lambda gt10 rabbit spleen cDNA library and sequenced. As predicted from the open reading frame (ORF) in vitro translation of a sense orientation GAPDH cDNA clone yielded a protein product with a molecular mass of 37 kDa. Rabbit GAPDH exhibits a high degree of homology to the mouse, rat, hamster, chicken and human GAPDH on both the nucleotide (nt) and amino acid (aa) levels.

The class of immunoglobulin is defined by the constant region of its heavy chain. When a B lymphocyte switches the class of heavy chain it produces, the constant region of mu-type heavy chain is replaced; this occurs through a DNA rearrangement that brings the gene segment encoding the new constant region close to the VDJ segment encoding the variable region. The pre-B-cell line 18-81, which switches from heavy chain mu to gamma 2b production in culture, occasionally abnormally rearranges the heavy chain locus so that DNA sequences between the switch regions of mu and gamma 2b are inverted. Because looping-out is an intermediate step in generating an inversion, the switch rearrangement could occur by looping-out and deletion. Provided that recombination is reciprocal, this would produce a circle of DNA. Indeed, circular DNA molecules have been isolated as products of rearrangement among gene segments encoding the variable regions of the T-cell receptor and of the immunoglobulin heavy chain and light chain. But whereas the breakpoints for the variable region rearrangement are precisely defined, the breakpoints for any given heavy chain class switch are scattered over a length of greater than 6 kilobases, including both switch regions. We have now isolated circular DNA containing the sequences deleted by class-switching, thereby showing that the immunoglobulin heavy chain class switch occurs through looping-out and deletion.

Immunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two light-chain loci: kappa and lambda. It has been reported that kappa loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged lambda-chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the kappa locus, and that only upon failure to produce a functional kappa chain is there an attempt to rearrange the lambda locus; and (b) the stochastic theory, which postulates that rearrangement at the lambda locus proceeds at a rate that is intrinsically much slower than that at the kappa locus. We show here that lambda-chain genes are generated whether or not the kappa locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.

The authors have developed a method to measure the rate of spontaneous mutations taking place in IgH, the gene encoding the immunoglobulin heavy chain. When an amber chain-termination codon mutates to a sense codon, translation of the polypeptide chain will be completed, and mutant cells producing the heavy chain can be detected with a fluorescent labelled antibody. The protocol used is the compartmentalization test which minimizes any effect of selection. In subclones of the pre-B lymphocyte line 18-81, the spontaneous mutation rate in the part of IgH encoding the variable region is somewhat greater than 10(-5) mutations per base pair per generation. This supports the hypothesis that hypermutation is not dependent on cell stimulation by an antigen. In a hybrid between a cell of this line and a myeloma (which represents the terminal stage of the B-cell lineage), the mutation rate was too low to be determined by this test, less than 10(-9). When the same loss to gain procedure system was used with an opal chain-terminating codon in the part of IgH encoding the constant region (C mu), a high rate of reversion by deletion was found. Long (more than one exon) and short (less than one exon) deletions occurred at rates of 1.7 x 10(-5) and 1.4 x 10(-7) per generation, respectively. It is thought that the high rate of deletion is not related to somatic hypermutation but rather to DNA rearrangement during the heavy-chain class switch, which is occurring in these pre-B cell lines. The point mutation rate was too low to be detected above the background of deletion mutants, less than 5 x 10(-8). The immunoglobulin mutator system works weakly, if at all, on two other, nonimmunoglobulin, genes tested: B2m (beta 2 microglobulin) and the gene for ouabain resistance.

Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.