Tumour Necrosis Factor alpha (TNF alpha), is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signalling events within cells, leading to necrosis or apoptosis The protein is also important for resistance to infection and cancers TNF alpha exerts many of its effects by binding, as a trimer, to either a 55 kDa cell membrane receptor termed TNFR-1 or a 75 kDa cell membrane receptor termed TNFR-2 Both these receptors belong to the so-called TNF receptor superfamily The superfamily includes FAS, CD40, CD27, and RANK The defining trait of these receptors is an extra cellular domain comprised of two to six repeats of cysteine rich motifs Additionally, a number of structurally related "decoy receptors" exist that act to sequester TNF molecules, thereby rescuing cells from apoptosis The crystal structures of TNF alpha, TNF beta, the extracellular domain of TNFR-1 (denoted sTNFR-1), and the TNF beta sTNFR-1 complex have been defined by crystallography This article will review the structure/function relationships of the TNF alpha and the TNF receptor superfamily It will also discuss insights as to how structural features play a role in the pleiotropic effects of TNF alpha

TNF alpha and the TNF receptor superfamily: structure-function relationship(s).

The lipocalin protein family: structural and sequence overview.

The present invention concerns fusion of Fc domains with biologically active peptides and a process for preparing pharmaceutical agents using biologically active peptides. In this invention, pharmacologically active compounds are prepared by a process comprising: a) selecting at least one peptide that modulates the activity of a protein of interest; and b) preparing a pharmacologic agent comprising an Fc domain covalently linked to at least one amino acid of the selected peptide. Linkage to the vehicle increases the half-life of the peptide, which otherwise would be quickly degraded in vivo. The preferred vehicle is an Fc domain. The peptide is preferably selected by phage display, E. coli display, ribosome display, RNA-peptide screening, or chemical-peptide screening.

Modified peptides as therapeutic agents

The TCL1 oncogene at 14q32.1 is involved in the development of human leukemia. This invention demonstrates the interaction between the Tcl1 and the Akt1 proteins. The physical interaction between endogenous Akt1 and Tcl1 occurs through the PH domain of the Akt1 protein. The present invention relates to the identification of Tcl1 mimics and Tcl1 antagonists that modifying this interaction, with the subsequent modification of apoptotic and proliferative signals.

https://www.pnas.org/content/pnas/97/7/3028.full.pdf

Tcl1 enhances akt kinase activity and mediates its nuclear translocation

Tick histamine-binding proteins: isolation, cloning, and three-dimensional structure.

Crystal structures have been determined of recombinant human tumor necrosis factor-alpha (TNF-alpha) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4(1)2(1)2 and had unit cell dimensions of a = b = 94.7 and c = 117.4 A. Refinement of the structure gave an R-factor of 22.3% at 2.5 A resolution. The crystals of TNF R31D mutant diffracted to 2.3 A resolution, and were of identical space group to the wild type with unit cell dimensions of a = b = 95.4 and c = 116.2 A, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 A for Calpha atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 A on Calpha atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.

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Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2.

Chromosomal rearrangements in the T cell malignancies observed in T cell prolymphocytic leukemias, and in patients with Ataxia telangiectasia involve the translocation of one T cell receptor gene to either chromosome 14q32 or Xq28 (1–3). These genetic aberrations juxtapose cellular protooncogenes to enhancer elements leading to deregulation of oncogene expression (4). The two oncogenes involved in these translocations are MTCP-1 and TCL-1, which constitute a family of genes involved in lymphoid proliferation and T cell malignancies (2, 5, 6).

The MTCP-1 (mature T cell proliferation-1) gene in the human X chromosome was the first candidate gene involved in the leukemogenesis of mature T cells (7). Aberrant or overexpressed MTCP-1 transcripts are found in the rare, but recurrent, chromosomal translocation t(X;14) in T cell proliferative diseases (8). About 10% of patients with the genetic disease Ataxia telangiectasia have clonal T cell proliferations with this cytogenetic aberration (1–3). MTCP-1 has two short ORFs that express A1 and B1 transcripts. The shorter A1 transcript is widely expressed in both tumor and nontumor cells. Expression of the B1 transcript is restricted to mature T cell proliferations with t(X;14) translocations (9), including T cell prolymphocytic leukemia tumors from patients with ataxia telangiectasia (10). The function of the B1 transcript of MTCP-1 is not known, but it encodes for a protein of 107 amino acids.

The TCL-1 oncogene maps at chromosome 14q32.1 (5). Chromosomal rearrangements at the TCL-1 locus are associated with T cell leukemia of the mature phenotype such as T cell prolymphocytic leukemia, adult T cell leukemia, and chronic T cell leukemias in patients with immunodeficiency syndrome Ataxia telangiectasia. TCL-1 is activated in T cell leukemias and lymphomas by chromosome translocations or inversions that juxtapose the α/δ or the β locus of the T cell receptor to the TCL-1 oncogene. TCL-1 is expressed at high levels in pre-B cells and immature thymocytes, but not in mature B or T cells (11). No expression of TCL-1 was observed in cell lines derived from normal human tissues. Ataxia telangiectasia patients with chromosome 14 rearrangements show expression of TCL-1 in leukemic T cells (10, 12). Similarly, the murine homolog of TCL-1 is expressed early in embryonic development in immature T and B cells (13).

The TCL-1 gene codes for a protein of 114 amino acid residues and molecular weight of 14,000 daltons (6). TCL-1 and MTCP-1 proteins share 40% identical amino acid residues and 61% similar amino acids, which strongly suggested that their tertiary structures are similar. However, no similarity was found with other human genes. Therefore, TCL-1 and MTCP-1 represent members of a unique family of genes involved in lymphoid proliferation and T cell malignancies. Human recombinant TCL-1 and MTCP-1 proteins have been expressed in Escherichia coli and purified for structural analysis to investigate their role in development of T cell malignancies (14). CD spectra at neutral pH are similar for both proteins and suggest β-sheet secondary structure. The crystal structure of MTCP-1 has been determined and refined to an R factor of 0.21 at 2.0 Å resolution. A model has been built of the structure of TCL-1. Analysis of these structures and the implications for the cellular function of these oncogene products are discussed.

https://www.pnas.org/content/pnas/95/7/3413.full.pdf

Crystal structure of MTCP-1: Implications for role of TCL-1 and MTCP-1 in T cell malignancies

The biological activities of tumor necrosis factor-alpha (TNF-alpha) are mediated by two different receptors, TNFR1 and TNFR2. To analyze the receptor binding site(s) of TNF-alpha, molecular models have been built of the complexes of TNF-alpha with the extracellular regions of receptors R1 and R2, based on the known crystal structures of TNF-alpha and lymphotoxin bound to R1. The model structure of R2 from residues 18-160 was built by analogy to the crystal structure of R1 in complex with lymphotoxin. The amino acid sequences of R1 and R2 show 27.5% identity over this region and were aligned with five insertions and three deletions. There are 18 conserved cysteines that form disulfides. R2 has lost one pair of cysteines compared with R1, but two new cysteines were modeled as forming a new disulfide bond. Both symmetric and asymmetric trimers of TNF-alpha were used to model the complexes with TNFR1 and R2. An analysis of differences in the model complexes showed good agreement with data on the differential binding of TNF mutants to its two receptors.

Z.-Q. Fu

Papers

Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2.

Crystal structure of MTCP-1: Implications for role of TCL-1 and MTCP-1 in T cell malignancies

Model complexes of tumor necrosis factor-alpha with receptors R1 and R2.

Crystal structure of mtcp-1 involved in t cell malignancies