Induction of cyclin D1 by simian virus 40 small tumor antigen

Cell-cycle progression is mediated by a coordinated interaction between cyclin-dependent kinases and their target proteins including the pRB and E2F/DP-1 complexes. Immunoneutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of the cyclin-dependent kinases, may be rate-limiting for G1 phase progression of the cell cycle. Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting G1 phase progression and augments substantially the efficiency of SV40 transformation through several distinct domains. In these studies, small t antigen stimulated cyclin D1 promoter activity 7-fold, primarily through an AP-1 binding site at −954 with additional contributions from a CRE site at −57. The cyclin D1 AP-1 and CRE sites were sufficient for activation by small t antigen when linked to an heterologous promoter. Point mutations of small t antigen between residues 97–103 that reduced PP2A binding were partially defective in the induction of the cyclin D1 promoter. These mutations also reduced activation of MEK1 and two distinct members of the mitogen-activated protein kinase family, the ERKs (extracellular signal regulated kinases) and the SAPKs (stress-activated protein kinases), in transfected cells. Dominant negative mutants of either MEK1, ERK or SEK1, reduced small t-dependent induction of the cyclin D1 promoter. SV40 small t induction of the cyclin D1 promoter involves both the ERK and SAPK pathways that together may contribute to the proliferative and transformation enhancing activity of small t antigen.

The kinetic mechanism of myosin V

Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590–593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin.

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.

The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T½ cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T½ derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.

An essential role of phosphatidylinositol 3-kinase in myogenic differentiation

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137), strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD, myosin heavy chain, creatine kinase, and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis.

Photoassembly of the manganese cluster and oxygen evolution from monomeric and dimeric CP47 reaction center photosystem II complexes

Isolated subcomplexes of photosystem II from spinach (CP47RC), composed of D1, D2, cytochrome b559, CP47, and a number of hydrophobic small subunits but devoid of CP43 and the extrinsic proteins of the oxygen-evolving complex, were shown to reconstitute the Mn4Ca1Clx cluster of the water-splitting system and to evolve oxygen. The photoactivation process in CP47RC dimers proceeds by the same two-step mechanism as observed in PSII membranes and exhibits the same stoichiometry for Mn2+, but with a 10-fold lower affinity for Ca2+ and an increased susceptibility to photodamage. After the lower Ca2+ affinity and the 10-fold smaller absorption cross-section for photons in CP47 dimers is taken into account, the intrinsic rate constant for the rate-limiting calcium-dependent dark step is indistinguishable for the two systems. The monomeric form of CP47RC also showed capacity to photoactivate and catalyze water oxidation, but with lower activity than the dimeric form and increased susceptibility to photodamage. After optimization of the various parameters affecting the photoactivation process in dimeric CP47RC subcores, 18% of the complexes were functionally reconstituted and the quantum efficiency for oxygen production by reactivated centers approached 96% of that observed for reconstituted photosystem II-enriched membranes.

Functioning of the Drosophila integral U1/U2 protein Snf independent of U1 and U2 small nuclear ribonucleoprotein particles is revealed by snf+ gene dose effects

Snf, encoded by sans fille, is the Drosophila homolog of mammalian U1A and U2B′′ and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that we infer must be physically separate from Snf’s functioning within snRNPs. Sxl is a master switch gene that controls its own pre-mRNA splicing as well as splicing for subordinate switch genes that regulate sex determination and dosage compensation. Exploiting an unusual new set of mutant Sxl alleles in an in vivo assay, we show that Snf is rate-limiting for Sxl autoregulation when Sxl levels are low. In such situations, increasing either maternal or zygotic snf+ dose enhances the positive autoregulatory activity of Sxl for Sxl somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a60, has no effect. Increased snf+ enhances Sxl autoregulation even when U1-70k and SF3a60 are reduced by mutation to levels that, in the case of SF3a60, demonstrably interfere with Sxl autoregulation. The observation that increased snf+ does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a60 is additional evidence that snf+ dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function.

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

In a previous examination using natural all-RNA substrates that contained either a 5′-oxy or 5′-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2′-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5′-thio group (R11S) and (ii) the departure of the 5′ leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg2+ ions were replaced by Mn2+ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg2+ ions were replaced by Mn2+ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5′-oxygen, because a switch in metal ion specificity was not observed with the 5′-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.

Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet

The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an l-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, l-[1-13C]cysteine was given as a primed, constant i.v. infusion (3μmol⋅kg−1⋅h−1) for 6 h, and incorporation of label into whole blood GSH determined by GC/MS selected ion monitoring. The fractional synthesis rate (mean ± SD; day-1) of whole blood GSH was 0.65 ± 0.13 for the adequate diet and 0.49 ± 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 ± 243 and 1,216 ± 162 μM for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 ± 216 and 579 ± 135 μmol⋅liter−1⋅day−1, respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects.

Melanoma angiogenesis and metastasis modulated by ribozyme targeting of the secreted growth factor pleiotrophin

Clinical and experimental evidence suggests that spreading of malignant cells from a localized tumor (metastasis) is directly related to the number of microvessels in the primary tumor. This tumor angiogenesis is thought to be mediated by tumor-cell-derived growth factors. However, most tumor cells express a multitude of candidate angiogenesis factors and it is difficult to decipher which of these are rate-limiting factors in vivo. Herein we use ribozyme targeting of pleiotrophin (PTN) in metastatic human melanoma cells to assess the significance of this secreted growth factor for angiogenesis and metastasis. As a model we used human melanoma cells (1205LU) that express high levels of PTN and metastasize from subcutaneous tumors to the lungs of experimental animals. In these melanoma cells, we reduced PTN mRNA and growth factor activity by transfection with PTN-targeted ribozymes and generated cell lines expressing different levels of PTN. We found that the reduction of PTN does not affect growth of the melanoma cells in vitro. In nude mice, however, tumor growth and angiogenesis were decreased in parallel with the reduced PTN levels and apoptosis in the tumors was increased. Concomitantly, the metastatic spread of the tumors from the subcutaneous site to the lungs was prevented. These studies support a direct link between tumor angiogenesis and metastasis through a secreted growth factor and identify PTN as a candidate factor that may be rate-limiting for human melanoma metastasis.

Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the β-secretase site of both the wild-type and Swedish mutant β-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the β-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of β-secretase, which catalyzes the rate-limiting step of the in vivo production of the β-amyloid (Aβ) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S1′ subsite, which prefers small side chains such as Ala, Ser, and Asp.

Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn2+ or Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA−•QB−• + H+ → QA(QBH)−, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD ≤ 0.5 μM) and reduced the observed value of kAB(2) 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB(2) reaction. In native RCs, kAB(2) was previously shown to be rate-limited by electron transfer based on the dependence of kAB(2) on the driving force for electron transfer. Upon addition of Zn2+ or Cd2+, kAB(2) became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+•QAQB−• → DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB−• occurs.

V(D)J recombination is not activated by demethylation of the kappa locus

V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.

Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of QB to QBH2 share a common entry point

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, QB. This process involves the transfer of two protons from the aqueous solution to the protein-bound QB molecule. The second proton, H+(2), is supplied to QB by Glu-L212, an internal residue protonated in response to formation of QA− and QB−. In this work, the pathway for H+(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of QA (DQA→D+QA−) and QB (DQB→D+QB−), studied by using pH-sensitive dyes. The other was the electron transfer kAB(1) (QA−QB→QAQB−). At pH 8.5, binding of Zn2+, Cd2+, or Ni2+ reduced the rates of proton uptake upon QA− and QB− formation as well as kAB(1) by ≈an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D+QA− is formed 105-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212→Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn2+ binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H+(2) was thus identified to be the same as for the first proton H+(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Oocytes are a source of catecholamines in the primate ovary: Evidence for a cell–cell regulatory loop

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine β-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes—but not those from which the oocytes had been removed—responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a β-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced β-adrenoreceptor activation.