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.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Simulation of Drosophila circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and CLK.

A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Active water in protein-protein communication within the membrane: the case of SRII-HtrII signal relay.

We detect internal water molecules in a membrane-embedded receptor-transducer complex and demonstrate water structure changes during formation of the signaling state. Time-resolved FTIR spectroscopy reveals stimulus-induced repositioning of one or more structurally active water molecules to a significantly more hydrophobic environment in the signaling state of the sensory rhodopsin II (SRII)-transducer (HtrII) complex. These waters, distinct from bound water molecules within the SRII receptor, appear to be in the middle of the transmembrane interface region near the Tyr199(SRII)-Asn74(HtrII) hydrogen bond. We conclude that water potentially plays an important role in the SRII --> HtrII signal transfer mechanism in the membrane's hydrophobic core.

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.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Intracellular signalling by the insulin receptor

The insulin receptor transduces insulin's biological signal through the tyrosine kinase present in the receptor's B subunit. The activated insulin receptor kinase then phosphorylates a series of intracellular substrate including insulin receptor substrate 1 (IRS-1), which has been shown to be the pivotal substrate for insulin receptor signal transduction. The phosphorylated tyrosine residues in IRS-1 can bind and activate the downstream effectors, many of which are SH2 domain containing proteins such as phosphotidylinositol 3-kinase, growth factor binding protein 2, and SH2 phosphotyrosine phosphatase 2. Phosphorylated synthetic IRS-1 peptides with the corresponding sequences of the IRS-1 have been shown to associate and activate their respective SH2 domain containing proteins. Another important event happening during insulin binding with the insulin receptor is that the insulin receptor rapidly undergoes internalization. However, the insulin receptor signalling and the receptor endocytosis have been studied as two independent processes. The hypothesis of the present thesis is that the insulin receptor endocytosis is involved in insulin receptor signalling and signal termination. The results of the present investigation demonstrate that insulin receptors in the earliest stage of endocytosis contain significantly greater kinase activity towards IRS-1 peptides than the receptors localized at the plasma membrane, indicating that they are potentially more capable of transducing signals. On the other hand, insulin receptors in the middle and late stage of endocytosis lose their kinase activity, suggesting that insulin receptor kinase activity inactivation and signal termination might take place in the late phase of the insulin receptor internalization. In addition, this study also found that the increased insulin receptor kinase activity in the endosomes is related to the tyrosyl phosphorylation of the specific domains of the receptor's $\beta$ subunit. ^

REPSA: A method for determining the sequence specificity of DNA binding ligands

We developed a novel combinatorial method termed restriction endonuclease protection selection and amplification (REPSA) to identify consensus binding sites of DNA-binding ligands. REPSA uses a unique enzymatic selection based on the inhibition of cleavage by a type IIS restriction endonuclease, an enzyme that cleaves DNA at a site distal from its recognition sequence. Sequences bound by a ligand are protected from cleavage while unprotected sequences are cleaved. This enzymatic selection occurs in solution under mild conditions and is dependant only on the DNA-binding ability of the ligand. Thus, REPSA is useful for a broad range of ligands including all classes of DNA-binding ligands, weakly binding ligands, mixed populations of ligands, and unknown ligands. Here I describe REPSA and the application of this method to select the consensus DNA-binding sequences of three representative DNA-binding ligands; a nucleic acid (triplex-forming single-stranded DNA), a protein (the TATA-binding protein), and a small molecule (Distamycin A). These studies generated new information regarding the specificity of these ligands in addition to establishing their DNA-binding sequences. ^

MECHANISMS OF SPECIES DIFFERENCES IN BRAIN ACETYLCHOLINESTERASE SENSITIVITY TO ORGANOPHOSPHATE INHIBITION

In vitro incubation of acetylcholinesterase from brain tissue of several species with organophosphate compounds indicated that the concentrations required to inhibit 50% of acetylcholinesterase activity (IC(,50)) differed from species to species for the same compound (Murphy, et al., 1968; Andersen, et al., 1972, 1977 and 1978).^ The hypothesis that non-specific binding proteins (Lauwerys and Murphy, 1969a,b) exerts a protective effect on acetylcholinesterase, and thus cause the differences observed in IC(,50) studies was tested by a ('3)H-DFP binding experiment. It was found that differences in the amount of non-specific binding protein cannot explain the observed differences observed in IC(,50) studies.^ An alternative hypothesis, that acetylcholinesterase from different species have different affinities for binding and/or different rates of phosphorylation by organophosphate insecticides was tested by determining the apparent affinity constant (k(,a)) and apparent rate of phosphorylation (k(,p)). Kinetic studies indicated that acetylcholinesterases from different species have different sensitivities to inhibition by organophosphate insecticides, and the differences are due to different affinities for binding and/or different rates of phosphorylation by the same organophosphate compound.^ Studies of the spontaneous reactivation of acetylcholinesterase after inhibition by organophosphate insecticides also indicated that acetylcholinesterases from different species have different rates and extents of spontaneous reactivation. This further substantiates the hypothesis that acetylcholinesterases from different species have different kinetic characteristics with respect to organophosphate insecticides inhibition.^ Eleven paraoxon analogs were synthesized for a quantitative structure-activity relationship study. It was found that the electron-withdrawing power ((sigma)) and hydrophobicity ((PARAGR)) of the substituent are important in determining the anti-cholinesterase activity of paraoxon analogs. Thus, predictions of species differences in acetylcholinesterase sensitivities to paraoxon analogs can be made if the physicochemical parameters ((sigma) and (PARAGR)) of the substituents are known.^ In another approach, i.e. enzyme modeling, the sensitivity of rat brain acetylcholinesterase to organophosphate insecticides was used as the independent variable to predict the sensitivities of acetylcholinesterases from other species to the same compound. Regression equations were derived for each species based on nineteen organophosphate insecticides studied. It was found, that in addition to paraoxon analogs, this method is also applicable to other organophosphate compounds with wide variations in structure. Thus, the sensitivities of acetylcholinesterases from other species can also be predicted from the sensitivity of rat brain acetylcholinesterase. ^

New aspects of estrogen action in the endometrium: Reciprocal interplay with retinoic acid pathway and a novel estrogen-regulated gene

The uterine endometrium is a major target for the estrogen. However, the molecular basis of estrogen action in the endometrium is largely unknown. I have used two approaches to study the effects of estrogen on the endometrium. One approach involved the study of the interaction between estrogen and retinoic acid (RA) pathways in the endometrium. I have demonstrated that estrogen administration to rodents and estrogen replacement therapy (ERT) in postmenopausal women selectively induced the endometrial expression of retinaldehyde dehydrogenase II (RALDH2), a critical enzyme of RA biosynthesis. RALDH2 was expressed exclusively in the stromal cells, especially in the stroma adjacent to the luminal and glandular epithelia. The induction of RALDH2 by estrogen required estrogen receptor and occurred via a direct increase in RALDH2 transcription. Among the three RA receptors, estrogen selectively induced the expression of RARα. In parallel, estrogen also increased the utilization of all-trans retinol (the substrate for RA biosynthesis) and the expression of two RA-regulated marker genes, cellular retinoic acid binding protein II (CRABP2) and tissue transglutaminase (tTG) in the endometrium. Thus estrogen coordinately upregulated both the production and signaling of RA in both the rodent and human endometrium. This coordinate upregulation of RA system appeared to play a role in counterbalancing the stimulatory effects of estrogen on the endometrium, since the depletion of endogenous RA in mice led to an increase in estrogen-stimulated stromal proliferation and endometrial Akt phosphorylation. In addition, I have also used a systematic approach (DNA microarray) to categorize genes and pathways affected by the ERT in the endometrium of postmenopausal women and identified a novel estrogen-regulated gene EIG121. EIG121 was exclusively expressed in the glandular epithelial cells of the endometrium and induced by estrogen in vivo and in cultured cell lines. Compared with the normal endometrium, EIG121 was highly overexpressed in type 1 endometrial cancer, but profoundly suppressed in type 2 endometrial tumors. Taken together, these studies suggested that estrogen regulates the expression of many genes of both the pro-proliferative and anti-proliferative pathways and the abnormality of these pathways may increase the risks for estrogen-dependent endometrial hyperplasia and endometrial cancer. ^

The regulation of JAB1 and its role in herceptin resistance

The Jun activation domain-binding protein (JAB1) is a c-Jun co-activator and a member of the COP9 signalosome. Additionally, it has recently been named a key negative regulator of the cyclin-dependent kinase inhibitor, p27. JAB1 overexpression has been observed in breast cancer and correlates with low p27 levels as well as poor prognosis, yet the mechanism of JAB1 deregulation is unknown. Data from our laboratory suggest that constitutive transcriptional activation of the jab1 gene is responsible for JAB1 protein overexpression. Therefore, we hypothesized that overexpression of JAB1 in breast cancer can be attributed to increased transcriptional activity. To identify potential positive regulators of JAB1, we characterized the promoter and found a 128 bp region that was critical for jab1 transcriptional activation. Our studies show that two oncogenic transcription factors, C/EBPβ and STAT3, play an important role in modulating jab1 transcription. Further, we have identified jab1 as a direct target gene of the SRC/STAT3 pathway. These studies provide insight to the mechanism of JAB1 overexpression in breast cancer and open up possibilities for therapies to inhibit its expression. ^ The development of the humanized monoclonal antibody, Herceptin (trastuzumab) targeting the HER2 (ErbB2) receptor has provided promising treatment to patients with aggressive HER2 positive breast cancer. However, many patients are resistant to Herceptin and additional therapies are needed to overcome resistance. Recent findings indicate that one mechanism of resistance involves AKT phosphorylation and subsequent mislocalization of the cyclin dependent kinase inhibitor, p27. We examined whether JAB1 facilitated degradation of p27 may be another mechanism of resistance to Herceptin. Our studies show that overexpression of JAB1 inhibited Herceptin induced G1-arrest and p27 accumulation. Interestingly, increased JAB1 levels were observed in two BT-474 Herceptin resistant clones. Targeted silencing of JAB1 increased p27 protein levels, reinstated a G1 checkpoint, and reduced cellular proliferation in the resistant clones. Our studies have demonstrated that inhibition of JAB1 sensitizes Herceptin resistant cells to treatment. Therefore, inhibition of JAB1 could provide a novel method of sensitizing resistant tumors to Herceptin-induced tumor growth arrest. ^

The regulation of mTORC2 kinase activity and complex integrity

Growth factor signaling promotes anabolic processes via activation of the PI3K-Akt kinase cascade. Deregulation of the growth factor-dependent PI3K-Akt pathway was implicated in tumorigenesis. Akt is an essential serine/threonine protein kinase that controls multiple physiological functions such as cell growth, proliferation, and survival to maintain cellular homeostasis. Recently, the mammalian Target of Rapamycin Complex 2 (mTORC2) was identified as the main Akt Ser-473 kinase, and Ser-473 phosphorylation is required for Akt hyperactivation. However, the detailed mechanism of mTORC2 regulation in response to growth factor stimulation or cellular stresses is not well understood. In the first project, we studied the regulation of the mTORC2-Akt signaling under ER stress. We identified the inactivation of mTORC2 by glycogen synthase kinase-3β (GSK-3β). Under ER stress, the essential mTORC2 component, rictor, is phosphorylated by GSK-3β at Ser-1235. This phosphorylation event results in the inhibition of mTORC2 kinase activity by interrupting Akt binding to mTORC2. Blocking rictor Ser-1235 phosphorylation can attenuate the negative impacts of GSK-3β on mTORC2/Akt signaling and tumor growth. Thus, our work demonstrated that GSK-3β-mediated rictor Ser-1235 phosphorylation in response to ER stress interferes with Akt signaling by inhibiting mTORC2 kinase activity. In the second project, I investigated the regulation of the mTORC2 integrity. We found that basal mTOR kinase activity depends on ATP level, which is tightly regulated by cell metabolism. The ATP-sensitive mTOR kinase is required for SIN1 protein phosphorylation and stabilization. SIN1 is an indispensable subunit of mTORC2 and is required for the complex assembly and mTORC2 kinase activity. Our findings reveal that mTOR-mediated phosphorylation of SIN1 is critical for maintaining complex integrity by preventing SIN1 from lysosomal degradation. In sum, our findings verify two distinct mTORC2 regulatory mechanisms via its components rictor and SIN1. First, GSK-3β-mediated rictor Ser-1235 phosphorylation results in mTORC2 inactivation by interfering its substrate binding ability. Second, mTOR-mediated Ser-260 phosphorylation of SIN1 preserves its complex integrity. Thus, these two projects provide novel insights into the regulation of mTORC2.

Regulation of the {\it neu\/} oncogene by the GTG element binding proteins

A previous study in our lab has shown that the transforming neu oncogene ($neu\sp\*$) was able to initiate signals that lead to repression of the neu promoter activity. Further deletion mapping of the neu promoter identified that the GTG element (GGTGGGGGGG), located between $-$243 and $-$234 relative to the translation initiation codon, mediates such a repression effect. I have characterized the four major protein complexes that interact with this GTG element. In situ UV-crosslinking indicated that each complex contains proteins of different molecular weights. The slowest migrating complex (S) contain Sp1 or Sp1-related proteins, as indicated by the data that both have similar molecular weights, similar properties in two affinity chromatographies, and both are antigenically related in gel shift analysis. Methylation protection and interference experiments demonstrated these complexes bind to overlapping regions of the GTG element. Mutations within the GTG element that either abrogate or enhance complex S binding conferred on the neu promoter with lower activity, indicating that positive factors other than Sp1 family proteins also contribute to neu promoter activity. A mutated version (mutant 4) of the GTG element, which binds mainly the fastest migrating complex that contains a very small protein of 26-kDa, can repress transcription when fused to a heterologous promoter. Further deletion and mutation studies suggested that this GTG mutant and its binding protein(s) may cooperate with some DNA element within a heterologous promoter to lock the basal transcription machinery; such a repressor might also repress neu transcription by interfering with the DNA binding of other transactivators. Our results suggest that both positive and negative trans-acting factors converge their binding sites on the GTG element and confer combinatorial control on the neu gene expression. ^