The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.

Long-form leptin receptor (OB-R(L)) is a signal-transducing member of the cytokine receptor superfamily that is essential for mediating the effects of leptin on mammalian body weight homeostasis. At present, the range of transcriptional targets responsive to OB-R(L) activation, and consequently, the likely mediators of leptin action, remain undefined. In this report, we have used cDNA subtractive hybridization to identify transcripts induced by leptin in immortalized hypothalamic neurons expressing OB-R(L). Differential expression of the identified transcripts in these cells was confirmed by both array technology and Northern blotting. In situ hybridization studies indicate that these transcripts are expressed in the mouse central nervous system, including nuclei of the hypothalamus that coexpress OB-R(L). Comparative in situ analysis of slices of hypothalami generated from control and leptin-injected ob/ob mice demonstrates that a subset of the identified transcripts is induced in vivo after leptin injection. The potential role of the proteins encoded by these transcripts in mediating the effects of leptin on body weight and energy homeostasis are discussed.

A mutation in the tub gene leads to maturity-onset obesity, insulin resistance, and progressive retinal and cochlear degeneration in mice. tub is a member of a growing family of genes that encode proteins of unknown function that are remarkably conserved across species. The absence of obvious transmembrane domain(s) or signal sequence peptide motif(s) suggests that Tub is an intracellular protein. Additional sequence analysis revealed the presence of putative tyrosine phosphorylation motifs and Src homology 2 (SH2)-binding sites. Here we demonstrate that in CHO-IR cells, transfected Tub is phosphorylated on tyrosine in response to insulin and insulin-like growth factor-1 and that in PC12 cells, insulin but not EGF induced tyrosine phosphorylation of endogenous Tub.In vitro, Tub is phosphorylated by purified insulin receptor kinase as well as by Abl and JAK 2 but not by epidermal growth factor receptor and Src kinases. Furthermore, upon tyrosine phosphorylation, Tub associated selectively with the SH2 domains of Abl, Lck, and the C-terminal SH2 domain of phospholipase Cγ and insulin enhanced the association of Tub with endogenous phospholipase Cγ in CHO-IR cells. These data suggest that Tub may function as an adaptor protein linking the insulin receptor, and possibly other protein-tyrosine kinases, to SH2-containing proteins.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner’s gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand‐binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog‐allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross‐link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE‐binding epitopes.

OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified G --> T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc«RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphor- ylated Fc«RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gand bsubunits of Fc«RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc«RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gand bsubunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the g and b subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated thatSykexhibitsahigheraffinityforthephosphorylatedtyrosineactivationmotifofthe gsubunitthanforthat of the bsubunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gsubunit motif. These data suggest that Syk associates with the g subunit of the high-affinity receptor for immunoglobulin E through an interaction betweenthetandemSH2domainsofSykandthephosphorylatedtyrosineactivationmotifofthe gsubunitand that Syk may be the major signaling protein that binds to Fc«RI tyrosine activation motifs in RBL-2H3 cells. Engagement of the high-affinity immunoglobulin E (IgE) receptor (FcεRI) on the surface of mast cells activates intra- cellular signal transduction pathways leading to the exocytic releaseofmediatorsoftheallergicresponse,suchasvasoactive amines, cytokines, and lipid metabolites. The release of these mediators is associated with the activation of multiple bio- chemical responses, including the stimulation of (i) serine, threonine and tyrosine phosphorylation of cellular proteins; (ii) phosphoinositide hydrolysis and phosphorylation; (iii) phospholipaseactivity;(iv)anincreaseinintracellularcalcium;

The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of a-helices connects the two SH2 domains, producing apr interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.

The complete primary structure of Fel d I-2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and chain 2), Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the domestic cat (Felis domesticus). An inframe linker encoding polyhistidine has been added to the 5' ends of the Fel d I chains 1 and 2 cDNAs to facilitate purification using Ni2+ ion affinity chromatography. This method provides high yields in a single step of rchain 1 and rchain 2 of Fel d I with a > 90% level of purity. Polymerase chain reaction (PCR) methods were used to introduce a thrombin cleavage site (LVPR decreases GS) at the N-terminus of both chains. Thrombin cleavage of rchain 1 and rchain 2 followed by HPLC purification of the cleavage products allowed the isolation of each recombinant chain with only two additional residuals (GS) at the N-terminus of the native sequence. Amino acid sequencing analysis of the N-terminus and mass spectrometry of these polypeptides demonstrated that they are highly pure and full-length. Direct ELISA assays showed that IgE from cat-allergic patients binds to both rchain 1 and rchain 2 of Fel d I, demonstrating that both these chains contribute to the allergenicity of this heterodimeric protein. An examination of the reactivity of T cells derived from cat-allergic patients revealed that both polypeptide chains contribute to the T cell response to this allergen. Consequently, it is concluded that the immunological response to Fel d I is composed of a reaction at both the B and T cell level to each of the two chains that constitute the native allergen.

Recombinant proteins expressed in E. coli are often sequestered into inclusion bodies and require the use of denaturing agents in order to solubilize them. The recombinant form of Amb a I, the major allergen from short ragweed pollen, is one such protein. In some cases solubility can be maintained after the removal of the denaturing agent, particularly if the protein can be folded into its native conformation. However, not all proteins refold readily and after the removal of the denaturing agent the proteins will reaggregate and/or precipitate. In the case of Amb a I, the recombinant protein stays in solution at low concentrations but aggregates with itself and other proteins. The recombinant Amb a I is not expressed at high levels and may be toxic to E. coli. Therefore, isolation from a complex mixture of E. coli proteins was necessary. Monoclonal antibodies which recognize the denatured form of Amb a I were available, allowing for immunoaffinity purification. However, because the protein was not monomeric, this chromatographic technique did not provide an improvement in the purity level when run in normal buffer solutions. Analysis of one monoclonal antibody's stability to urea indicated it could tolerate the presence of 2 M urea and recover full activity. Use of this antibody as an immunoaffinity reagent in a column run in 2 M urea, which minimized aggregation of the E. coli produced proteins, gave a high degree of purification of recombinant Amb a I in one step. This illustrates the potential for the use of denaturing and other solubilizing agents in immunoaffinity chromatography of recombinant proteins.

The genes encoding chain 1 (Ch1) and chain 2 (Ch2) of the major allergen of the domestic cat, Felis domesticus I, have been analyzed by genomic cloning and by polymerase chain reaction (PCR). Ch1 has two potential leader sequences, designated A and B. Analysis of a genomic clone encoding Ch1 demonstrated that one structural gene contains sequences corresponding to both leaders, which utilize different Met start codons. PCR analysis showed that genes encoding Ch1 and Ch2 are co-expressed in both the salivary glands and the skin, and that leader sequence A of Ch1 is utilized preferentially in both tissues. Ch2 was shown to have two dominant forms that are differentially expressed in the aforementioned tissues. The long form (Ch2L), composed of 92 amino acids (aa), is preferentially expressed in the salivary glands, while the short form (Ch2S), composed of 90 aa, is preferentially expressed in the skin. There is minor sequence polymorphism in both forms of Ch2. A genomic clone for Ch2 only contained sequences for Ch2S, suggesting that Ch2L is encoded by an exon not contained within this genomic clone.