Allosteric activation of VCP, a AAA unfoldase, by small molecule mimicry.

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme activity, activator binding must be permissive to different conformational states needed for enzyme function. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to ∼3-fold. Our cryo-EM studies resulted in structures (∼2.9-3.5 Å-resolution) of VCP in apo and ADP-bound states, and revealed VA1 binding an allosteric pocket near the C-terminus in both states. Engineered mutations in the VA1 binding site confer resistance to VA1, and furthermore, modulate VCP activity to a similar level as VA1-mediated activation. The VA1 binding site can alternatively be occupied by a phenylalanine residue in the VCP C-terminal tail, a motif that is post-translationally modified and interacts with cofactors. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry. Significance: The loss of function of valosin-containing protein (VCP/p97), a mechanoenzyme from the AAA superfamily that hydrolyzes ATP and uses the released energy to extract or unfold substrate proteins, is linked to protein aggregation-based disorders. However, druggable allosteric sites to activate VCP, or any AAA mechanoenzyme, have not been identified. Here, we report cryo-EM structures of VCP in two states in complex with VA1, a compound we identified that dose-dependently stimulates VCP's ATP hydrolysis activity. The VA1 binding site can also be occupied by a phenylalanine residue in the VCP C-terminal tail, suggesting that VA1 acts through mimicry of this interaction. Our study reveals a druggable allosteric site and a mechanism of enzyme regulation.

Pelvic connective tissue resilience decreases with vaginal delivery, menopause and uterine prolapse.

BACKGROUND: The late onset of pelvic visceral prolapse and incontinence after childbirth injury could be explained by menopause-associated connective tissue weakening. Uterosacral ligament resilience (UsR) was assessed to determine whether it influenced uterine or pelvic floor mobility, or varied with age, vaginal delivery, menopause or histological variations in the ligament. METHODS: UsR was measured by tensiometry in ligaments from 85 hysterectomy specimens, and was correlated with the presence of symptomatic uterocervical prolapse, prehysterectomy uterine and anorectal mobility, patient age, history of vaginal delivery and menopause. Forty-five of these ligaments were examined for ligament thickness, muscle to collagen ratio, and oestrogen and progesterone receptor density. The results were correlated with UsR. RESULTS: UsR was significantly reduced (P = 0.02) in symptomatic uterovaginal prolapse, but there was no correlation with either uterocervical or anorectal descent in women without symptomatic prolapse. There was a significant decrease in UsR with vaginal delivery (P = 0.003), menopause (P = 0.009) and older age (P = 0.005). The uterosacral ligament was significantly thinner and contained fewer oestrogen and progesterone receptors after menopause, but this did not affect UsR. CONCLUSION: Where pelvic floor muscles are weakened, decreases in pelvic connective tissue resilience related to the menopause may facilitate progression to symptomatic pelvic visceral prolapse.

Evidence for coherent proton tunneling in a hydrogen bond network.

We observed coherent proton tunneling in the cyclic network of four hydrogen bonds in calix[4]arene. The tunneling frequency of 35 megahertz was revealed by a peak in the magnetic field dependence of the proton spin-lattice relaxation rate measured with field-cycling nuclear magnetic resonance in the solid state at temperatures below 80 kelvin. The amplitude of the coherent tunneling peak grows with temperature according to a Boltzmann law with energy D/kB = (125 +/- 10) kelvin (where kB is Boltzmann's constant). The tunneling peak can be interpreted in the context of level crossings in the region where the tunneling frequency matches the proton Larmor frequency. The tunneling spectrum reveals fine structure that we attribute to coupling between the hydrogen bonds in the network. The characteristics of the tunneling peak are interpreted in the context of the potential energy surface experienced by the hydrogen atoms in the network.

Treatment of cat allergy with T-cell reactive peptides.

We induced in allergic humans the counterpart of murine experimental T-cell tolerance. T-cell lines from cat-allergic humans were used to map T-cell epitopes for the principal allergen of cat dander, Fel d 1. Two peptides of 27 amino acids each were synthesized to contain the dominant epitopes (ALLERVAX CAT). After a safety trial, we carried out a blinded study of the dose required for efficacy. We randomly divided 95 cat-sensitive patients into placebo, 7.5 micrograms, 75 micrograms, and 750 micrograms groups. Patients received a subcutaneous injection weekly for 4 wk. Before and after treatment, patients were exposed in a room inhabited by live cats and scored by nose and lung symptoms. Baseline nasal and lung scores (+/-SEM) were 6.2 +/- 0.56 and 5.4 +/- 0.73 in the 750 micrograms group; 7.8 +/- 0.53 and 4.7 +/- 0.68 in the placebo group. Six weeks after treatment, scores adjusted for baseline differences were reduced in the 750 micrograms group: -2.3 +/- 4.9 and -2.3 +/- 0.59 compared with -0.84 +/- 0.50 and -0.85 +/- 0.62 in the placebo group. The 75 micrograms group showed intermediate effects and the 7.5 micrograms group no effect. Linear trend analysis indicated a significant dose response effect: p = 0.05 for nose and 0.03 for lung symptoms. Allergic side effects occurred an hour or more after the first 750 micrograms dose in 16 of 24 patients but required little or no treatment with one exception. T-cell reactive treatment peptides safely improved allergic responses to cats.

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Expression and function of a CD5 cDNA in human and murine T cells.

In order to define the function of the CD 5 (T1, Leu 1, Tp 67 in the human; Ly-1 in the mouse) molecule, a cDNA clone of human CD 5 was expressed in a CD5-deficient Jurkat cell line and in a murine T cell hybridoma. A Jurkat subclone (Jurkat 9.9) produced interleukin 2 (IL 2) in response to anti-CD 3 monoclonal antibody (mAb) cross-linked to solid support. IL 2 production was enhanced by co-culture with the anti-CD 5 mAb OKT 1. A CD 5-deficient mutant clone Jurkat 1.15 was generated by treatment with ethyl methanesulfonate followed by selection with anti-CD 5 mAb plus complement. Jurkat 1.15 did not demonstrate enhancement of IL 2 production by OKT 1 in the presence of cross-linked anti-CD 3 mAb. A cDNA encoding human CD 5 was introduced into a defective retrovirus which was used to infect Jurkat 1.15. A Jurkat clone stably expressing CD 5 was established. In response to OKT 1, a rise in intracellular calcium was observed in both the parent Jurkat 9.9 and the CD 5+ infectant but not in the CD 5- mutant or a G 418- resistant control. Furthermore, expression of CD 5 restored the augmentation of IL 2 production by OKT 1 in response to cross-linked anti-CD 3 mAb. A murine T cell hybridoma By 155.16 which produces IL 2 in response to HLA-DR antigens was also infected with the CD 5-recombinant retrovirus and three stable CD 5+ infectants were generated. These hybridomas showed enhancement of IL 2 production by stimulation with OKT 1 in the presence of suboptimal concentrations of soluble anti-murine CD 3 mAb. These results provide further evidence that CD 5 provides a co-stimulatory signal for T cell activation.

Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth.

Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a lambda gt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggests that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution.

An evaluation of an enzymatic choline determination for the identification of semen in casework samples.

This study compares the detection of choline in seminal stains by both an enzymatic method and by the standard Florence crystal test. The tests were conducted on 293 actual casework samples. In those samples identified as containing semen, choline was detected twice as often by the enzymatic method compared to the Florence method (84.6 versus 40.3%). The choline results were correlated with spermatozoa and acid phosphatase tests. The enzymatic detection of choline in seminal stains was found to be a fast, easy, sensitive, and reliable test.

Molecular cloning of Ly-1, a membrane glycoprotein of mouse T lymphocytes and a subset of B cells: molecular homology to its human counterpart Leu-1/T1 (CD5).

We report the isolation of cDNA clones of the mouse lymphocyte differentiation antigen Ly-1. One of these cDNA clones was confirmed to be full-length by DNA sequencing and by expression of Ly-1 by L cells transfected with this clone. Analysis of the predicted amino acid sequence indicated that the Ly-1 polypeptide is synthesized with a 23 amino acid leader and that the mature protein consists of an amino-terminal region of 347 amino acids, a transmembrane sequence of 30 residues, and a carboxyl-terminal region of 94 amino acids. The amino-terminal region appears to be divided into two subregions by a threonine- and proline-rich sequence of 23 amino acids that is highly conserved between Ly-1 and its human homologue Leu-1 (CD5) in position and amino acid composition. The first amino-terminal subregion of 111 amino acids is predicted to be arranged in a beta-pleated sheet structure of six strands. The entire amino-terminal region is rich in cysteine, with all of its 22 cysteine residues conserved between Ly-1 and Leu-1. The carboxyl-terminal region has no cysteines. Ly-1 and Leu-1 are 63% identical, with a gradient of identical residues from 43% for the first amino-terminal subregion to 58% for the second amino-terminal subregion and 90% for the carboxyl-terminal region. The predicted secondary structure of the first amino-terminal subregion and identities of certain conserved residues among most members of the immunoglobulin gene superfamily suggest that Ly-1 and Leu-1 are distant members of this family.

Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T1/Leu-1.

The T1/Leu-1/CD5 molecule, a human T-cell surface glycoprotein of relative molecular mass (Mr) 67,000, has been implicated in the proliferative response of activated T cells and in T-cell helper function. A similar involvement in T-cell proliferation has been reported for Ly-1, the murine homologue of T1. Here we report the complete amino-acid sequence of the T1 precursor molecule deduced from complementary DNA clones. The protein contains a classical signal peptide; a 347-amino-acid extracellular segment; a transmembrane region; and a 93-amino-acid intracellular segment. The extracellular segment contains many cysteine residues and is composed of two related structural domains separated by a proline/threonine-rich region. The T1 molecule has structural features characteristic of other receptor molecules.

Characterization and distribution of a 24,000-molecular weight antigen defined by a monoclonal antibody (DU-ALL-1) elicited to common acute lymphoblastic leukemia (cALL) cells.

A monoclonal antibody (DU-ALL-1) was generated to common acute lymphoblastic leukemia (cALL) cells by microcytotoxicity and indirect immunofluorescence, DU-ALL-1 reacted only with cALL cell lines and not with the other hematopoietic cell lines tested. Peripheral blood lymphocytes, monocytes, granulocytes and mitogen-activated lymphocytes did not react significantly with this antibody. However, platelets (100%) and normal bone marrow cells (8.5%) reacted with DU-ALL-1. Microcytotoxicity testing of human leukemia cells showed that DU-ALL-1 reacted with cells from a majority of null and pre-B ALL patients (63/77) and with cells from some patients with acute myeloblastic leukemia (4/7) and T-ALL (4/20). DU-ALL-1 was generally non-reactive with cells from patients with B-cell leukemias (2/16) and chronic myelogenous leukemia in blast crisis (0/4). By an indirect immunoperoxidase technique, DU-ALL-1 reacted with a variety of non-hematopoietic tissues, including smooth and cardiac muscle and epithelia from several organs. The DU-ALL-1 antigen had an apparent mol. wt of 24,000 and did not bind to lectins or label with [3H]glucosamine. Thus, DU-ALL-1 defines a 24,000-mol. wt protein which is absent from most peripheral blood mononuclear cells, is expressed on normal platelets and several non-hematopoietic tissues, and may be a useful for subclassifying leukemias.

Classification of human leukemia by membrane antigen analysis with xenoantisera.

Rabbit and monkey antisera after appropriate absorption were rendered specific for normal or leukemic lymphoid- and myeloid-associated antigens. Antisera defining a common peripheral blood T-cell antigen, a thymus leukemia antigen, HLA-DR or Ia-like antigen, common acute lymphoblastic leukemia antigen (CALLA), and a myeloid-monocyte (M) antigen were used in a microcytotoxicity assay to classify leukemic cells from 30 patients in a double blind study. The antisera to the M antigen reacted with adherent peripheral blood cells and polymorphonuclear leukocytes and failed to react with nonadherent mononuclear cells and enriched T-cells and chronic lymphocytic leukemia cells. The M antisera also reacted with U937, a monocytic-type cell line, and with HL60, a promyelocytic-type cell line, but failed to react with T and B lymphoblastoid cell lines. The specificities of the other antisera have been described in previous reports. Cells from three of the patients could not be phenotyped by microcytotoxicity testing. Cells from 25 patients had a consensus morphological or histochemical diagnosis of either acute lymphoblastic leukemia or acute nonlymphocytic leukemia. The serological classification of these patients using the five types of antisera listed above were consistent with the consensus diagnosis. In addition, the lymphoid cancers were further subclassified as to T-, B-, or thymus antigen types. There was no consensus lymphoid versus myeloid diagnosis on cells from two patient. The serological classification in both cases favored a diagnosis of myeloid rather than lymphoid leukemia.

Phenotypic characterization of human bone marrow granulocyte-macrophage forming progenitor cells.

Cell surface antigens of the human bone marrow CFU-C have been studied. Human marrow cells were incubated with a variety of monoclonal antisera and complement prior to culture in semisolid media. By using indirect immunofluorescent studies, the percentage of bone marrow cells binding the antibodies was determined. The CFU-C phenotype is HLA+, la+, 4F2+, 3A1-, and DUALL-1-. This study provides information that is useful in the study of myeloid cell ontogeny and necessary for the use of some of these reagents in the treatment of bone marrow cells prior to human bone marrow transplantation in various clinical settings.

Distribution of common acute lymphoblastic leukemia antigen in nonhematopoietic tissues.

The common acute lymphoblastic leukemia antigen (CALLA), as defined by J-5 murine monoclonal antibodies, was detected on renal tubular and glomerular cells from fetal and adult donors by an indirect immunoperoxidase technique. CALLA could also be detected on epithelial cells of the fetal small intestine and on myoepithelial cells of adult breast but not on myoepithelial cells of the salivary gland. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated 125I-labeled membrane antigens from dissociated renal cells demonstrated that the antigen migrated as a 90,000 mol wt antigen rather than the 98,000-100,000 mol wt antigen noted on CALLA-positive tissue culture cell lines. The data suggest that the determinant defined by the J-5 monoclonal antibody is neither a lymphoid cell-specific differentiation antigen nor a leukemia-specific antigen.