Mutations affecting beta-tubulin folding and degradation.

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

Expression and hormonal regulation of Muc-1 at the apical surface of mouse uterine epithelial cells and its role in modulating embryo implantation

Previous studies from our lab have established that large molecular weight mucin glycoproteins are major apically-disposed components of mouse uterine epithelial cells in vitro (Valdizan et al., (1992) J. Cell. Physiol. 151:451-465). The present studies demonstrate that Muc-1 represents one of the apically-disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and mRNA expression are regulated in the peri-implantation stage mouse uterus by ovarian steroids. Muc-1 expression is high in the proestrous and estrous stages, and decreases during diestrous. Both Muc-1 protein and mRNA levels decline to barely detectable levels by day 4 of pregnancy, i.e., prior to the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by administration of exogenous progesterone. Initiation of implantation was triggered by coinjection of progesterone maintained mice with a nidatory dose of 17$\beta$-estradiol. Muc-1 levels in the uterine epithelia of progesterone maintained mice declined to similar low levels as observed on day 4 of normal pregnancy. Coinjection of estradiol did not alter Muc-1 expression suggesting that down-regulation of Muc-1 is a progesterone dominated event. This was confirmed in ovariectomized, non-pregnant mice which displayed stimulation of Muc-1 expression following 6 hr of estradiol injection. Estradiol stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. While progesterone alone had no effect on Muc-1 expression, it antagonized estradiol action in this regard. Injection of pregnant mice with the antiprogestin, RU 486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is progesterone receptor-mediated. Muc-1 appears to function as an anti-adhesive molecule at the apical cell surface of mouse uterine epithelial cells. Treatment of polarized cultures of mouse uterine epithelial cells with O-sialoglycoprotein endopeptidase reduced mucin expression in vitro, by about 50%, and converted polarized uterine epithelia to a functionally receptive state. Similarly, ablation of Muc-1 in Muc-1 null mice resulted in polarized uterine epithelia that were functionally receptive as compared to their wild-type counterparts in vitro. Collectively, these data indicate that Muc-1 and other mucins function as anti-adhesive molecules and that reduction or removal of these molecules is a prerequisite for the generation of a receptive uterine state. ^

In vitro release of dimethyloxaloylglycine and l-mimosine from bovine bone mineral.

OBJECTIVE Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes. MATERIALS Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and (3)[H]thymidine incorporation, respectively. RESULTS We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed. CONCLUSIONS In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration.

L-mimosine and dimethyloxaloylglycine decrease plasminogen activation in periodontal fibroblasts

BACKGROUND The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium. METHODS Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction. RESULTS The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1-stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed. CONCLUSION L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.

Dimethyloxalylglycine lyophilized onto bone substitutes increase vessel area in rat calvarial defects.

AIM Pharmacological inhibitors of prolyl hydroxylases, also termed hypoxia-mimetic agents (HMAs), when repeatedly injected can support angiogenesis and bone regeneration. However, the possible role of HMA loaded onto bone substitutes to support angiogenesis and bone regeneration under diabetic condition is unknown. The capacity of HMA loaded onto deproteinized bovine bone mineral (DBBM) to support angiogenesis and bone formation was examined in diabetic Wistar rats. METHODS Diabetes was induced by intraperitoneal injection of streptozotocin. The HMA dimethyloxalylglycine (DMOG) and desferrioxamine (DFO) were lyophilized onto DBBM. Calvarial defects were created with a trephine drill and filled with the respective bone substitutes. After 4 weeks of healing, the animals were subjected to histological and histomorphometric analysis. RESULTS In this report, we provide evidence that DMOG loaded onto DBBM can support angiogenesis in vivo. Specifically, we show that DMOG increased the vessel area in the defect site to 2.4% ± 1.3% compared with controls 1.1% ± 0.48% (P = 0.012). There was a trend toward an increased vessel number in the defect site with 38.6 ± 17.4 and 31.0 ± 10.3 in the DMOG and the control group (P = 0.231). The increase in angiogenesis, however, did not translate into enhanced bone formation in the defect area with 9.2% ± 7.1% and 8.4% ± 5.6% in DMOG and control group, respectively. No significant changes were caused by DFO. CONCLUSIONS The results suggest that DMOG loaded onto DBBM can support angiogenesis, but bone formation does not increase accordingly in a type 1 diabetic rat calvarial defect model at the indicated time point.

L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

AIM To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. METHODOLOGY Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. RESULTS Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P < 0.05). This effect on VEGF was reduced by echinomycin (P < 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P > 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P < 0.05) and IL-8 levels (P < 0.05). CONCLUSIONS The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy.

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.

A chloroplast processing enzyme functions as the general stromal processing peptidase

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

Funding: Wellcome Trust, 070247/Z/03/A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Transfer of the HIV-1 cyclophilin-binding site to simian immunodeficiency virus from Macaca mulatta can confer both cyclosporin sensitivity and cyclosporin dependence

HIV-1 specifically incorporates the peptidyl prolyl isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). HIV-1 replication is inhibited by CsA as well as by nonimmunosuppressive CsA analogues that bind to CyPA and interfere with its virion association. In contrast, the related simian immunodeficiency virus SIVmac, which does not interact with CyPA, is resistant to these compounds. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction between the active site of the enzyme and the capsid (CA) domain of the HIV-1 Gag polyprotein. We report here that the transfer of HIV-1 CA residues 86–93, which form part of an exposed loop, to the corresponding position in SIVmac resulted in the efficient incorporation of CyPA and conferred an HIV-1-like sensitivity to a nonimmunosuppressive cyclosporin. HIV-1 CA residues 86–90 were also sufficient to transfer the ability to efficiently incorporate CyPA, provided that the length of the CyPA-binding loop was preserved. However, the resulting SIVmac mutant required the presence of cyclosporin for efficient virus replication. The results indicate that the presence or absence of a type II tight turn adjacent to the primary CyPA-binding site determines whether CyPA incorporation enhances or inhibits viral replication. By demonstrating that CyPA-binding-site residues can induce cyclosporin sensitivity in a heterologous context, this study provides direct in vivo evidence that the exposed loop between helices IV and V of HIV-1 CA not merely constitutes a docking site for CyPA but is a functional target of this cellular protein.

All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae

The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown. Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding. We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae. Five cyclophilins and three FKBPs previously were identified in yeast. We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3. None of the eight cyclophilins or four FKBPs were essential. Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation. We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes. We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.

Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures

Trigger factor (TF) in Escherichia coli is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL’s affinity for unfolded proteins, and promotes degradation of certain polypeptides. Because the latter effects appeared larger at 20°C, we studied the influence of temperature on TF expression. Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42°C to 16°C and even rose in cells stored at 4°C. Upon temperature downshift from 37°C to 10°C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins. We therefore tested if TF expression might be important for viability at low temperatures. When stored at 4°C, E. coli lose viability at exponential rates. Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability. Although TF overproduction protected against cold, it reduced viability at 50°C, while TF deficiency enhanced viability at this temperature. By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4°C, which may explain why expression of hsps is suppressed in the cold. Thus, TF represents an example of an E. coli protein which protects cells against low temperatures. Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes.

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.

Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo in Saccharomyces cerevisiae

Cyclophilin and FK506 binding protein (FKBP) accelerate cis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.