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Dysregulated immune responses are essential underlying causes of a plethora of pathologies including cancer, autoimmunity, and immunodeficiency. We here investigated 4 patients from unrelated families presenting with immunodeficiency, autoimmunity, and malignancy. We identified 4 distinct homozygous mutations in TNFRSF9 encoding the tumor necrosis factor receptor superfamily member CD137/4-1BB, leading to reduced, or loss of, protein expression. Lymphocytic responses crucial for immune surveillance, including activation, proliferation, and differentiation, were impaired. Genetic reconstitution of CD137 reversed these defects. CD137 deficiency is a novel inborn error of human immunity characterized by lymphocytic defects with early-onset Epstein-Barr virus (EBV)-associated lymphoma. Our findings elucidate a functional role and relevance of CD137 in human immune homeostasis and antitumor responses., (© 2019 by The American Society of Hematology.)

Cellular responses to signals require the action of a myriad of protein networks, which are regulated by protein/protein associations. Rapid actions of steroid hormones are also subject to this regulation. They induce direct association of steroid receptors with different proteins (e.g., growth factor receptors, signaling effectors, scaffold proteins, transcription factors). These multi-molecular complexes drive signaling activation and finally trigger basic hormonal effects. Receptor/protein associations are attracting increased interest concerning their role in hormone action as well as their potential use as therapeutic targets in hormonal diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER., (Published by Elsevier B.V.)

The tumor suppressor p53 and the DNA repair factor 53BP1 (p53 binding protein 1) regulate gene transcription and responses to genotoxic stresses. Upon DNA damage, p53 undergoes dimethylation at Lys382 (p53K382me2), and this posttranslational modification is recognized by 53BP1. The molecular mechanism of nonhistone methyl-lysine mark recognition remains unknown. Here we report a 1. 6-A-resolution crystal structure of the tandem Tudor domain of human 53BP1 bound to a p53K382me2 peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation-pi interactions in a cage formed by four aromatic residues and an aspartate of 53BP1. The signature HKKme2 motif of p53, which defines specificity, is identified through a combination of NMR resonance perturbations, mutagenesis, measurements of binding affinities and docking simulations, and analysis of the crystal structures of 53BP1 bound to p53 peptides containing other dimethyl-lysine marks, p53K370me2 (p53 dimethylated at Lys370) and p53K372me2 (p53 dimethylated at Lys372). Binding of the 53BP1 Tudor domain to p53K382me2 may facilitate p53 accumulation at DNA damage sites and promote DNA repair as suggested by chromatin immunoprecipitation and DNA repair assays. Together, our data detail the molecular mechanism of p53-53BP1 association and provide the basis for deciphering the role of this interaction in the regulation of p53 and 53BP1 functions., ((c) 2010 Elsevier Ltd. All rights reserved.)

The keystone of the adaptive immune response is T cell receptor (TCR) recognition of peptide presented by major histocompatibility complex (pMHC) molecules. The crystal structure of AHIII TCR bound to MHC, HLA-A2, showed a large interface with an atypical binding orientation. MHC mutations in the interface of the proteins were tested for changes in TCR recognition. From the range of responses observed, three representative HLA-A2 mutants, T163A, W167A, and K66A, were selected for further study. Binding constants and co-crystal structures of the AHIII TCR and the three mutants were determined. K66 in HLA-A2 makes contacts with both peptide and TCR, and has been identified as a critical residue for recognition by numerous TCR. The K66A mutation resulted in the lowest AHIII T cell response and the lowest binding affinity, which suggests that the T cell response may correlate with affinity. Importantly, the K66A mutation does not affect the conformation of the peptide. The change in affinity appears to be due to a loss in hydrogen bonds in the interface as a result of a conformational change in the TCR complementarity-determining region 3 (CDR3) loop. Isothermal titration calorimetry confirmed the loss of hydrogen bonding by a large loss in enthalpy. Our findings are inconsistent with the notion that the CDR1 and CDR2 loops of the TCR are responsible for MHC restriction, while the CDR3 loops interact solely with the peptide. Instead, we present here an MHC mutation that does not change the conformation of the peptide, yet results in an altered conformation of a CDR3.

Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here, we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20, 46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 1981, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets., (Copyright 2003 Elsevier B.V.)

In unstressed cells, the tumor suppressor protein p53, a tetrameric transcription factor, is present in a latent state and is maintained at low levels through targeted degradation. A variety of cellular stresses including DNA damage, hypoxia, nucleotide depletion, viral infection, and cytokine-activated signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G1/S or G2/M transitions of the cell cycle or to apoptosis. The molecular mechanisms by which stabilization and activation occur are incompletely understood, but accumulating evidence points to roles for multiple posttranslational modifications in mediating these events through several potentially interacting but distinct pathways. Both the approximately 100 amino acid N-terminal and approximately 90 amino acid C-terminal domains are highly modified by phosphorylation and acetylation, whereas modifications to the central sequence-specific DNA binding domain have not been reported. Seven serines and one threonine in the first 46 residues of the transactivation domain and four to five serines in the carboxyl-terminal domain are now known to be phosphorylated, and Lys320 and Lys382 in the carboxyl-terminal domain (human p53) can be acetylated. Antibodies that recognize p53 only when it has been modified at specific sites have been developed by several laboratories, and studies with these have shown that most of the known posttranslational modifications are induced when cells are exposed to DNA-damaging agents. Exceptions are Ser378, which is reported to be constitutively phosphorylated, and Ser376, which is dephosphorylated in response to DNA damage. These recent results, coupled with biochemical and genetic studies, suggest that several amino-terminal phosphorylations can be important in stabilizing p53 in response to DNA damage and in directing acetylation at C-terminal sites. DNA damage-induced modifications to the C-terminus inhibit the ability of this domain to negatively regulate sequence-specific DNA binding either by inducing a conformational change in the protein or by inhibiting non-sequence-specific DNA binding by the C-terminus. C-terminal modifications also modulate the oligomerization state of p53, and may modulate nuclear import/export. Modifications in response to DNA damage to other components that interact with p53 may also be important. In most cases, clear roles for specific modifications, interactions among individual modifications, and the enzymes responsible for each modification remain to be defined. Nevertheless, the field appears poised for major advances in the understanding of the molecular mechanisms that regulate p53 function.

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

The C-terminal domain of the tumor suppressor protein p53 is the site of non-specific DNA binding. Purified p53 produced from baculovirus-infected insect cells binds preferentially to supercoiled DNA, forming bands with lower electrophoretic mobility. This non-covalent binding does not change the linking number of the DNA. An anti-p53 antibody targeting the C-terminal domain partially competes with supercoiled DNA in binding to p53, while antibodies targeted to the N terminus of p53 supershift the complex bands. A synthetic peptide corresponding to amino acid residues 319-393 of human p53 also displays preferential binding to supercoiled DNA, while a mutant peptide, which is unable to form tetramers, is inactive. The center of the equilibrium distribution of topoisomers formed by relaxation with topoisomerase I is not shifted in the presence of p53 although the distribution is broadened. The preferential binding of p53 is exhibited toward both positively and negatively supercoiled DNA. These observations are consistent with a model in which p53 binds to right-handed or left-handed strand crossings.

The major site of interaction between MHC class II molecules and invariant chain has been mapped to occupancy of the class II peptide-binding site by the CLIP region of invariant chain. CLIP is also seen as a degradation product of invariant chain and can be found in association with class II as a processing intermediate. Here we analyzed the relative contribution of single amino acids in the murine CLIP (86-102) peptide for binding to I-Ab and I-Ad and for recognition by a CLIP-specific T cell hybridoma. Interestingly, the I-Ab-restricted murine T cell hybridoma that recognizes murine CLIP peptide (86-102) is dependent on Met 102 for activation. This amino acid is outside of the core binding region and in the CLIP/DR3 crystal structure extends outside of the class II peptide-binding site. These data suggest that a T cell epitope presented on CLIP/class II complexes can be located predominantly in flanking residues that extend out of the peptide binding groove of class II., (Copyright 1998 Academic Press.)

The principal neutralizing determinant of the human immunodeficiency virus type 1 (HIV-1) is an intra-chain disulphide-bridged loop, designated V3, in the third hypervariable region of the surface glycoprotein gp120. Peptide sequences from the V3 loop of gp120 from HIV-1 strain MN (HIV-1MN) were engineered into the N-terminal region of the major coat protein of filamentous bacteriophage fd, leading to their display in multiple copies on the surface of the bacteriophage virion. Peptides displayed in this way were shown to be remarkably effective structural mimics of the natural epitope. They were recognised by human HIV antisera and evoked high titres of antibodies in mice, which cross-reacted with other strains of HIV and were capable of neutralizing the virus. In addition, antibody production could be stimulated by simultaneous inoculation with T-cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins and is a promising vaccine model.

The formation of peptide/major histocompatibility complex (MHC) complexes and their subsequent recognition by T cells is a pivotal event in the initiation of an immune response. While X-ray crystal structures are now available for class I MHC/peptide complexes, little detailed structural information is known about the class II MHC equivalent, and there are no solution structure data for either. A 16 amino acid residue moth cytochrome c peptide (residues 88 to 103) was 13C-labeled for two-dimensional isotope-edited NMR analysis. The peptide was labeled either selectively in the methyl groups of alanine residues or uniformly at every carbon position, and bound to unlabeled soluble mouse I-Ek class II MHC molecules. Although alpha-helical in the native cytochrome c protein and with no uniform structure in solution, the peptide is bound to the I-Ek molecule with the alpha-carbon atoms of the 11 C-terminal residues held in the binding groove. This indicates that the class II MHC peptide binding site is somewhat larger than that of class I MHC molecules (> or = 11 amino acid residues versus 8 to 10 amino acid residues), consistent with recent data on eluted peptides. Despite the large size of the complex (approximately 70 kDa), nuclear Overhauser effects are clearly detectable between peptide side-chains and the MHC molecule. Indications of the buried or exposed nature of particular side-chains within the bound peptide are derived from the NMR data and these are used together with information from previous biological studies to propose a crude model of the interaction of the peptide with the groove of the MHC molecule. We find no evidence for a conformational change in the peptide/MHC complex in the spectra at pH 5.0 versus pH 7.0, despite a 40-fold faster on-rate for the peptide at the lower pH value.

The three-dimensional structure of a 43-residue active, synthetic peptide encompassing the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of a multi-cooling dynamical simulated annealing protocol using restraints derived from 1H nuclear magnetic resonance spectroscopy. A total of 442 experimentally derived restraints including 13 dihedral angle (phi, chi 1) restraints were used. A final set of 35 structures was calculated with a root-mean-square deviation from the mean co-ordinates of 0.36 A for the backbone atoms and 0.96 A when side-chain heavy atoms were included for the well-defined region comprising residues Val7 to Leu39. Although assignments were made and sequential connectivities observed for the N-terminal six and C-terminal four residues, the absence of long-range NOEs suggests that the terminal regions are largely unstructured. The binding domain contains two short parallel alpha-helices (residues Val7 to Lys14 and Lys32 to Leu39), a3(10)-helix (residues Asp17 to Val21) and a structured loop made up of overlapping beta-turns (residues Gln22 to Leu31), which enclose a close-packed hydrophobic core. The loop is stabilized to a large extent by Asp34. This residue is conserved in all peripheral subunit-binding domains and its carboxylate side-chain forms a set of side-chain-main-chain hydrogen bonds with the main-chain amide protons of Gly23, Thr24, Gly25 and Leu31 and a side-chain-side-chain hydrogen bond with the hydroxyl group of Thr24. We propose that a peripheral subunit-binding site may be located in the loop region, which contains a series of highly conserved residues and provides a number of potential recognition sites. The structured region of the binding domain, comprising 33 residues, represents an exceptionally short amino acid sequence with defined tertiary structure that has no disulphide bond, ligand or cofactor to stabilize the fold. It may be approaching the lower size limit for a three-dimensional structure possessing features characteristic of larger structures, including a close-packed, non-polar interior. The organization of the side-chains in the hydrophobic core may have implications for de novo protein design.

The three-dimensional structure of a DNA tridecamer d(CGCAGAATTCGCG)2 containing bulged adenine bases was determined by single crystal X-ray diffraction methods, at 120 K, to 2.6 A resolution. The structure is a B-DNA type double helix with a single duplex in the asymmetric unit. One of the bulged adenine bases loops out from the double helix, while the other stacks in to it. This is in contrast to our preliminary finding, which indicated that both adenine bases were looped out. This revised model was confirmed by the use of a covalently bound heavy-atom derivative. The conformation of the looped-out bulge hardly disrupts base stacking interactions of the bases flanking it. This is achieved by the backbone making a "loop-the-loop" curve with the extra adenine flipping over with respect to the other nucleotides in the strand. The looped-out base intercalates into the stacked-in bulge site of a symmetrically related duplex. The looped-out and stacked-in bases form an A.A reversed Hoogsteen base-pair that stacks between the surrounding base-pairs, thus stabilizing both bulges. The double helix is frayed at one end with the two "melted" bases participating in intermolecular interactions. A related structure, of the same tridecamer, after soaking the crystals with proflavin, was determined to 3.2 A resolution. The main features of this B-DNA duplex are basically similar to the native tridecamer but differ in detail especially in the conformation of the bulged-out base. Accommodation of a large perturbation such as that described here with minimal disruption of the double helix shows both the flexibility and resiliency of the DNA molecule.

In human plasma, apolipoprotein A-I is present as pro and mature isoproteins. The development of a highly specific antibody to the pro isoprotein of apoA-I has been difficult due to the close structural similarity between the pro and mature isoforms of apoA-I. To sermount this difficulty, a peptide was synthesized by the solid phase method which corresponded to the amino acid sequence present in the pro region of apoA-I. The synthetic peptide was coupled to serum albumin and the conjugate utilized to immunize rabbits for antibody production. Immunoblot analysis of purified proapoA-I and mature apoA-I revealed that the antibody was specific for the propeptide of apoA-I. Analysis of apoA-I in the plasma from a Tangier disease patient and newly secreted apoA-I from HepG2 cells clearly demonstrated the isoforms which contained the proisoprotein. The proapoA-I specific antibody should prove to be a useful tool in developing a radioimmunoassay for quantitation of the proisoprotein in plasma, isolation of proapoA-I from normal and dyslipoproteinemic subjects by immunoaffinity chromatography and in studies related to the synthesis and processing of apoA-I.

Crystals of an almost self-complementary DNA 15-mer d(CGCGAAATTTACGCG) have been grown by the vapor diffusion technique at 4 degrees C. The space group is I222 with a = 37.3 A, b = 54.6 A and c = 104.8 A. Solution studies showed that the 15-mer forms a duplex with the extra adenine residue unpaired: (sequence; see text) Crystals are stable at 4 degrees C and are suitable for medium-resolution structural studies.

The hen egg-white lysozyme (HEL)-specific suppression induced by soluble molecules produced by a monoclonal T-cell lymphoma line (LH8-105) obtained from HEL-specific suppressor T lymphocytes has been examined. Injection of I-J+ molecules from LH8-105 cell culture supernatant (TsFa) in HEL-primed mice during the afferent phase of the response induced Lyt-2+ second order suppressor T (Ts) cells which, upon transfer into HEL-CFA-primed syngeneic recipients, inhibit the delayed-type hypersensitivity (DTH) response to HEL. Transfer of spleen cells from TsFa-injected mice primed with HEL or human lysozyme suppresses the DTH response to HEL in recipient mice whereas this response is not affected by cell transfer from ring-necked pheasant egg-white lysozyme (REL)-primed and TsFa-injected mice, indicating that induction of second order Ts by TsFa is specific for a lysozyme epitope including phenylalanine at position 3. Fine antigenic specificity of second order Ts-cell induction is confirmed by similar results obtained upon injection of TsFa in mice primed with HEL N-terminal synthetic peptide or with an analog in which, as in REL, phenylalanine has been substituted by tyrosine at position 3. The same fine antigenic specificity observed in the induction of second order Ts cells is also present in the expression of TsFe suppressive activity. The similar antigenic specificity of Tsa and Tse suggests that Tse cells could result from amplification of the Tsa cell population or these two cell subsets could reflect different maturation stages of the same cell type rather than distinct T-cell populations activated in cascade.

Crystals of the DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G) have been grown by the vapor-diffusion technique with 2-methyl-2,4-pentanediol as precipitant. They are monoclinic space group C2, with a = 79.6 A, b = 43.1 A, c = 24.9 A and beta = 98.7 degrees. Previous nuclear magnetic resonance studies predicted that this tridecamer forms a duplex similar to the B DNA dodecamer, d(C-G-C-G-A-A-T-T-C-G-C-G), except for an extra adenosine residue that is stacked within the helix but remains unpaired: (formula; see text) Preliminary X-ray diffraction studies confirmed that the tridecamer is in the B DNA conformation, consistent with the nuclear magnetic resonance results.