Gene-experience interaction alters the cholinergic septohippocampal pathway of mice

Spatial learning requires the septohippocampal pathway. The interaction of learning experience with gene products to modulate the function of a pathway may underlie use-dependent plasticity. The regulated release of nerve growth factor (NGF) from hippocampal cultures and hippocampus, as well as its actions on cholinergic septal neurons, suggest it as a candidate protein to interact with a learning experience. A method was used to evaluate NGF gene-experience interaction on the septohippocampal neural circuitry in mice. The method permits brain region-specific expression of a new gene by using a two-component approach: a virus vector directing expression of cre recombinase; and transgenic mice carrying genomic recombination substrates rendered transcriptionally inactive by a “floxed” stop cassette. Cre recombinase vector delivery into transgenic mouse hippocampus resulted in recombination in 30% of infected cells and the expression of a new gene in those cells. To examine the interaction of the NGF gene and experience, adult mice carrying a NGF transgene with a floxed stop cassette (NGFXAT) received a cre recombinase vector to produce localized unilateral hippocampal NGF gene expression, so-called “activated” mice. Activated and control nonactivated NGFXAT mice were subjected to different experiences: repeated spatial learning, repeated rote performance, or standard vivarium housing. Latency, the time to complete the learning task, declined in the repeated spatial learning groups. The measurement of interaction between NGF gene expression and experience on the septohippocampal circuitry was assessed by counting retrogradely labeled basal forebrain cholinergic neurons projecting to the hippocampal site of NGF gene activation. Comparison of all NGF activated groups revealed a graded effect of experience on the septohippocampal pathway, with the largest change occurring in activated mice provided with repeated learning experience. These data demonstrate that plasticity of the adult spatial learning circuitry can be robustly modulated by experience-dependent interactions with a specific hippocampal gene product.

Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced β-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21–26 nt) corresponding to the 3′ proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3′ end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage

DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD+, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3Cdc34 goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

Abnormal transport along the lysosomal pathway in Mucolipidosis, type IV disease

Mucolipidosis, type IV (ML-IV) is an autosomal recessive storage disease that is characterized by lysosomal accumulation of sphingolipids, phospholipids, and acid mucopolysaccharides. Unlike most other storage diseases, the lysosomal hydrolases participating in the catabolism of the stored molecules appear to be normal. In the present study, we examined the hypothesis that the ML-IV phenotype might arise from abnormal transport along the lysosomal pathway. By using various markers for endocytosis, we found that plasma membrane internalization and recycling were nearly identical in ML-IV and normal fibroblasts. A fluorescent analog of lactosylceramide (LacCer) was used to study plasma membrane lipid internalization and subsequent transport. Lipid internalization at 19°C was similar in both cell types; however, 40–60 min after raising the temperature to 37°C, the fluorescent lipid accumulated in the lysosomes of ML-IV cells but was mainly concentrated at the Golgi complex of normal fibroblasts. Biochemical studies demonstrated that at these time points, hydrolysis of the lipid analog was minimal (∼7%) in both cell types. A fluorescence ratio imaging assay was developed to monitor accumulation of fluorescent LacCer in the lysosomes and showed that the apparent concentration of the lipid increased more rapidly and to a greater extent in ML-IV cells than in normal fibroblasts. By 60 min, LacCer apparently decreased in the lysosomes of normal fibroblasts but not in ML-IV cells, suggesting that lipid efflux from the lysosomes was also impaired. These results demonstrate that there is a defect in ML-IV fibroblasts that affects membrane sorting and/or late steps of endocytosis.

Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: The phenylacetyl-CoA catabolon

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a β-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor β superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E2 (PGE2) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE2 within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE2 and cyclooxygenase inhibitors on this process. PGE2 can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE2 to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE2-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte–somatic cell interactions in female reproduction.

The adaptor protein Crk connects multiple cellular stimuli to the JNK signaling pathway

c-Jun N-terminal kinases (JNKs) are potently activated by a number of cellular stimuli. Small GTPases, in particular Rac, are responsible for initiating the activation of the JNK pathways. So far, the signals leading from extracellular stimuli to the activation of Rac have remained elusive. Recent studies have demonstrated that the Src homology 2 (SH2)- and Src homology 3 (SH3)-containing adaptor protein Crk is capable of activating JNK when ectopically expressed. We found here that transient expression of Crk induces JNK activation, and this activation was dependent on both the SH2- and SH3-domains of Crk. Expression of p130Cas (Cas), a major binding protein for the Crk SH2-domain, also induced JNK activation, which was blocked by the SH2-mutant of Crk. JNK activation by Cas and Crk was effectively blocked by a dominant-negative form of Rac, suggesting for a linear pathway from the Cas-Crk-complex to the Rac-JNK activation. Many of the stimuli that activate the Rac-JNK pathway enhance engagement of the Crk SH2-domain. JNK activation by these stimuli, such as epidermal growth factor, integrin ligand binding and v-Src, was efficiently blocked by dominant-negative mutants of Crk. A dominant-negative form of Cas in turn blocked the integrin-, but not epidermal growth factor - nor v-Src-mediated JNK activation. Together, these results demonstrate an important role for Crk in connecting multiple cellular stimuli to the Rac-JNK pathway, and a role for the Cas-Crk complex in integrin-mediated JNK activation.

Metabolism to a response pathway selective retinoid ligand during axial pattern formation

We report identification of 9-cis-4-oxo-retinoic acid (9-cis-4-oxo-RA) as an in vivo retinoid metabolite in Xenopus embryos. 9-Cis-4-oxo-RA bound receptors (RARs) α, β, and γ as well as retinoid X receptors (RXRs) α, β, and γ in vitro. However, this retinoid displayed differential RXR activation depending on the response pathway used. Although it failed to activate RXRs in RXR homodimers, it activated RXRs and RARs synergistically in RAR-RXR heterodimers. 9-Cis-4-oxo-RA thus acted as a dimer-specific agonist. Considering that RAR-RXR heterodimers are major functional units involved in transducing retinoid signals during embryogenesis and that 9-cis-4-oxo-RA displayed high potency for modulating axial pattern formation in Xenopus, metabolism to 9-cis-4-oxo-RA may provide a mechanism to target retinoid action to this and other RAR-RXR heterodimer-mediated processes.

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.

Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: Description of the folding pathway

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of 104 structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix α1 (residues 13–17) and in helix α2 as well as a turn and nonnative hydrophobic clustering between β3 and β4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in α1 and the C-terminal portion of the β-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from φ value analysis and NMR.

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.

ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.