Extended kalman filter for estimation of parameters in nonlinear state-space models of biochemical networks.

It is system dynamics that determines the function of cells, tissues and organisms. To develop mathematical models and estimate their parameters are an essential issue for studying dynamic behaviors of biological systems which include metabolic networks, genetic regulatory networks and signal transduction pathways, under perturbation of external stimuli. In general, biological dynamic systems are partially observed. Therefore, a natural way to model dynamic biological systems is to employ nonlinear state-space equations. Although statistical methods for parameter estimation of linear models in biological dynamic systems have been developed intensively in the recent years, the estimation of both states and parameters of nonlinear dynamic systems remains a challenging task. In this report, we apply extended Kalman Filter (EKF) to the estimation of both states and parameters of nonlinear state-space models. To evaluate the performance of the EKF for parameter estimation, we apply the EKF to a simulation dataset and two real datasets: JAK-STAT signal transduction pathway and Ras/Raf/MEK/ERK signaling transduction pathways datasets. The preliminary results show that EKF can accurately estimate the parameters and predict states in nonlinear state-space equations for modeling dynamic biochemical networks.

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

14-3-3 proteins interact with the beta-thymosin repeat protein Csp24.

Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.

Phosphorylation in the regulation of muscle-specific transcription

The contents of this dissertation include studies on the mechanisms by which FGF and growth factor down-stream kinases inactivate myogenin; characterization of myogenin phosphorylation and its role in regulation of myogenin activity; analysis the C-terminal transcriptional activation domain of myogenin; studies on the nuclear localization of myogenin and characterization of proteins that interact with PKC.^ Activation of muscle transcription by the MyoD family requires their heterodimerization with ubiquitous bHLH proteins such as the E2A gene products E12 and E47. I have shown that dimerization with E2A products potentiates phosphorylation of myogenin at serine 43 in its amino-terminus and serine 170 in the carboxyl-terminal transcription activation domains. Mutations of these sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. Consistent with the role of phosphorylation at serine 170, analysis of the carboxyl-terminal transcriptional activation domain by deletion has revealed a stretch of residues from 157 to 170 which functions as a negative element for myogenin activity.^ In addition to inducing phosphorylation of myogenin, E12 also localizes myogenin to the nucleus. The DNA binding and dimerization mutants of myogenin show various deficiencies in nuclear localization. Cotransfection of E12 with the DNA binding mutants, but not a dimerization mutant, greatly enhances their nuclear binding. These data suggest that the nuclear localization signal is located in the DNA binding region and myogenin can also be nuclear localized by virtue of dimerizing with a nuclear protein.^ FGF is one of the most potent inhibitors of myogenesis and activates many down-stream pathways to exert its functions. One of these pathway is the MAP kinase pathway. Studies have shown that Raf-1 and Erk-1 kinase inactivate transactivation by myogenin and E proteins independent of DNA binding. The other is the PKC pathway. In transfected cells, FGF induces phosphorylation of thr-87 that maps to the previously identified PKC sites in the DNA binding domain of myogenin. Myogenin mutant T-N87 could resist the inhibition directed to the bHLH domain by FGF, suggesting that FGF inactivates myogenin by inducing phosphorylation of this site. In C2 myotubes, where FGF receptors are lost, the phosphatase inhibitor, okadaic acid, and phorbal ester PdBu, can also induce the phosphorylation of thr-87. This result supports the previous observation and suggests that in myotubes, other mechanisms, such as innervation, may inactivate myogenin through PKC induced phosphorylation.^ Many functions of PKC have been well documented, yet, little is known about the activators or effectors of PKC or proteins that mediate PKC nuclear localizations. Identification of PKC binding proteins will help to understand the molecular mechanism of PKC function. Two proteins that interact with the C kinase (PICKS) have been characterized, PICK-1 and PICK-2. PICK1 interacts with two conserved regions in the catalytic domain of PKC. It is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK2 is a novel protein with homology to the heat shock protein family. It interacts extensively with the catalytic domain of PKC and is localized in the cytoplasm in a punctate pattern. PICK1 and PICK2 may play important roles in mediating the actions of PKC. ^

Identification of the signal pathways modulated by expression of p210$\rm\sp{bcr-abl}$ and the inhibition of p210$\rm\sp{bcr-abl}$ transforming activity by polypeptides interacting with p210$\rm\sp{bcr-abl}$ in murine myeloid 32D cells

The Bcr-Abl fusion oncogene which resulted from a balanced reciprocal translocation between chromosome 9 and 22, t(9;22)(q11, q34), encodes a 210 KD elevated tyrosine specific protein kinase that is found in more than 95 percent of chronic myelogenous leukemia patients (CML). Increase of level of phosphorylation of tyrosine is observed on cell cycle regulatory proteins in cells overexpressing the Bcr-Abl oncogene, which activates multiple signaling pathways. In addition, distinct signals are required for transforming susceptible fibroblast and hematopoietic cells, and the minimal signals essential for transforming hematopoietic cells are yet to be defined. In the present study, we first established a tetracycline repressible p210$\rm\sp{bcr-abl}$ expression system in a murine myeloid cell line 32D c13, which depends on IL3 to grow in the presence of tetracycline and proliferate independent of IL3 in the absence of tetracycline. Interestingly, one of these sublines does not form tumors in athymic nude mice suggesting that these cells may not be completely transformed. These cells also exhibit a dose-dependent growth and expression of p210$\rm\sp{bcr-abl}$ at varying concentrations of tetracycline in the culture. However, p210$\rm\sp{bcr-abl}$ rescues IL3 deprivation induced apoptosis in a non-dose dependent fashion. DNA genotoxic damage induced by gamma-irradiation activates c-Abl tyrosine kinase, the cellular homologue of p210$\rm\sp{bcr-abl},$ and leads to activation of p38 MAP kinase in the cells. However, in the presence of p210$\rm\sp{bcr-abl}$ the irradiation failed to activate the p38 MAP kinase as examined by an antibody against phosphorylated p38 MAP kinase. Similarly, an altered tyrosine phosphorylation of the JAK1-STAT1 pathways was identified in cells constitutively overexpressing p210$\rm\sp{bcr-abl}.$ This may provided a molecular mechanism for altered therapeutic response of CML patients to IFN-$\alpha.$^ Bcr-Abl oncoprotein has multiple functional domains which have been identified by the work of others. The Bcr tetramerization domain, which may function to stabilize the association of the Bcr-Abl with actin filaments in p210$\rm\sp{bcr-abl}$ susceptible cells, are essential for transforming both fibroblast and hematopoietic cells. We designed a transcription unit encoding first 160 amino acids polypeptide of Bcr protein to test if this polypeptide can inhibit the transforming activity of the p210$\rm\sp{bcr-abl}$ oncoprotein in the 32D c13 cells. When this vector was transfected transiently along with the p210$\rm\sp{bcr-abl}$ expression vector, it can block the transforming activity of p210$\rm\sp{bcr-abl}.$ On the other hand, the retinoblastoma tumor suppressor protein (Rb), a naturally occurring negative regulator of the c-Abl kinase, the cellular homologue of Bcr-Abl oncoprotein, binds to and inhibits the c-Abl kinase in a cell cycle dependent manner. A polypeptide obtained from the carboxyl terminal end of the retinoblastoma tumor suppressor protein, in which the nuclear localization signal was mutated, was used to inhibit the kinase activity of the p210$\rm\sp{bcr-abl}$ in the cytoplasm. This polypeptide, called Rb MC-box, and its wild type form, Rb C-box, when overexpressed in the 32D cells are mainly localized in the cytoplasm. Cotransfection of a plasmid transcription unit coding for this polypeptide and the gene for the p210$\rm\sp{bcr-abl}$ resulted in reduced plating efficiency of p210$\rm\sp{bcr-abl}$ transfected IL3 independent 32D cells. Together, these results may lead to a molecular approach to therapy of CML and an in vitro assay system to identify new targets to which an inhibitory polypeptide transcription unit may be directed. ^

Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.

BACKGROUND It is unknown why patients with extensive ulcerative colitis (UC) have a higher risk of colorectal cancer compared with patients with left-sided UC. This study characterizes the inflammatory processes in left-sided UC, pancolitis, and UC-associated dysplasia at the transcriptional level to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. METHODS The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction and immunohistochemistry data by the Wilcoxon's rank-sum test. RESULTS The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel upregulated and downregulated transcripts, respectively, were identified in specimens from dysplasia and pancolitis. Validation of selected transcripts hereof identified insulin receptor alpha (INSRA) and MAP kinase interacting serine/threonine kinase 2 (MKNK2) with an enhanced expression in dysplasia compared with left-sided UC and controls, whereas laminin γ2 (LAMC2) was found with a lower expression in dysplasia compared with the remaining 3 groups. CONCLUSIONS This study demonstrates pancolitis and left-sided UC as distinct inflammatory processes at the transcriptional level, and identifies INSRA, MKNK2, and LAMC2 as potential critical transcripts in the inflammation-driven preneoplastic process of UC.

Frequency modulation of ERK activation dynamics rewires cell fate.

Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.

Growth cone MKK7 mRNA targeting regulates MAP1b-dependent microtubule bundling to control neurite elongation

Local mRNA translation in neurons has been mostly studied during axon guidance and synapse formation but not during initial neurite outgrowth. We performed a genome-wide screen for neurite-enriched mRNAs and identified an mRNA that encodes mitogen-activated protein kinase kinase 7 (MKK7), a MAP kinase kinase (MAPKK) for Jun kinase (JNK). We show that MKK7 mRNA localizes to the growth cone where it has the potential to be translated. MKK7 is then specifically phosphorylated in the neurite shaft, where it is part of a MAP kinase signaling module consisting of dual leucine zipper kinase (DLK), MKK7, and JNK1. This triggers Map1b phosphorylation to regulate microtubule bundling leading to neurite elongation. We propose a model in which MKK7 mRNA localization and translation in the growth cone allows for a mechanism to position JNK signaling in the neurite shaft and to specifically link it to regulation of microtubule bundling. At the same time, this uncouples activated JNK from its functions relevant to nuclear translocation and transcriptional activation.

Interference by the intracellular parasite Theileria parva with T-cell signal transduction pathways induces transformation and protection against apoptosis

The intracellular parasite Theileria parva transforms bovine T-lymphocytes, inducing uncontrolled proliferation. Upon infection, cells cease to require antigenic stimulation and exogenous growth factors to proliferate. Earlier studies have shown that pathways triggered via stimulation of the T-cell receptor are silent in transformed cells. This is reflected by a lack of phosphorylation of key signalling molecules and the fact that proliferation is not inhibited by immunosuppressants such as cyclosporin and ascomycin that target calcineurin. This suggests that the parasite bypasses the normal T-cells activation pathways to induce proliferation. Among the MAP-kinase pathways, ERK and p38 are silent, and only Jun N-terminal kinase is activated. This appears to suffice to induce constitutive activation of the transcription factor AP-1. More recently, it could be shown that the presence of the parasite in the host cell cytoplasm also induces constitutive activation of NF-kappaB, a transcription factor involved in proliferation and protection against apoptosis. Activation is effectuated by parasite-induced degradation of IkappaBs, the cytoplasmic inhibitors which sequester NF-kappaB in the cytoplasm. NF-kappaB activation is resistant to the antioxidant N-acetyl cysteine and a range of other reagents, suggesting that activation might occur in an unorthodox manner. Studies using inhibitors and dominant negative mutants demonstrate that the parasite activates a NF-kappaB-dependent anti-apoptotic mechanism that protects the transformed cell form spontaneous apoptosis and is essential for maintaining the transformed state of the parasitised cell.

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

NRAS and BRAF mutations in primary cutaneous melanoma: A comparison of mutation rates between radial and vertical growth phases in individual tumors

Primary cutaneous melanoma is a cancer arising from melanocytes in the skin. In recent decades the incidence of this malignancy has increased significantly. Mortality rates are high for patients with tumors measuring over a few millimeters in thickness. Response rates to conventional radiation and chemotherapy are very low in patients with metastatic melanoma. New therapies targeting melanoma’s aberrant cell signaling pathways such as the MAP Kinase pathway are being developed. Mutations of NRAS and BRAF genes are quite common in cutaneous melanoma and lead to constitutive activation of the MAP Kinase pathway. This study tests the hypothesis that NRAS and BRAF mutations increase as a tumor progresses from the noninvasive radial growth phase (RGP) to the invasive vertical growth phase (VGP). Laser capture microdissection was used to obtain separate, pure tumor DNA samples from the RGP and VGP of thirty primary cutaneous melanomas. PCR was used to amplify NRAS exon 2 and BRAF exon 15 tumor DNA. The amplified DNA was sequenced and analyzed for mutations. An overall mutation rate of 74% was obtained for the twenty-three melanomas in which there were complete sequence results. With the exception of one melanoma NRAS and BRAF mutations were mutually exclusive. All seven NRAS exon 2 mutations involved codon 61. Three of these melanomas had mutations in both the RGP and VGP. The remaining four tumors were wild type for NRAS exon 2 in the RGP but mutated in the VGP. Of the fifteen BRAF exon 15 mutated melanomas all but one involved codon 600. Twelve of the fifteen BRAF exon 15 mutations were the T1799A type. Nine of the fifteen BRAF mutated tumors had the same mutation in both the RGP and VGP. Five of fifteen melanomas had wild type RGP DNA and BRAF exon 15 mutated VGP DNA. A single melanoma had BRAF exon 15 mutated DNA in the RGP and wild type DNA in the VGP. Overall, these results suggest a trend toward the acquisition of NRAS and BRAF mutations as cutaneous melanomas change from a noninvasive to an invasive, potentially deadly cancer.^

Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor κ B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-κB, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.

The effect of protein tyrosine phosphatase SHP1 on tumorigenicity of the MDA -MB231 breast cancer cells

SHP1 is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. It is highly expressed in hematopoietic cells and expressed in normal epithelium at lower levels. While SHP1 in hematopoietic cells is thought to be a negative regulator of cellular signaling by associating with and dephosphorylating various receptors and their downstream effectors after they become activated, its precise function in epithelium remains to be understood. The potential involvement of SHP1 in human tumorigenesis has been hypothesized from the findings that SHP1 can interact with, dephosphorylate, and regulate the activity of several protein tyrosine kinases (PTKs) implicated in human cancer. These PTKs include epidermal growth factor receptor (EGFR) and Src. Such speculation is also supported by the report that SHP1 is overexpressed in human ovarian cancers. ^ Here we report, for the first time, that the levels of SHP1 expression and activity are altered in human breast cancer cells in comparison with normal breast epithelium. In particular, SHP1 expression is nearly lost in the breast cancer cell lines MDA-MB231 and MDA-MB435. After the re-introduction of SHP1 both in wild type (wt) and enzymatically inactive (dn) forms, into the MDA-MB231 cells, we observed no changes in cellular proliferation. However, the overexpression of wt SHP1 led to increased anchorage-independent growth in the MDA-MB231 cells. SHP1 phosphatase activity is essential for such an increase since the overexpression of dn SHP1 had no effect. Enhanced turnorigenicity in nude mice was also observed in the MDA-MB231 cells overexpressing wt SHP1, but not dn SHP1, suggesting the crucial function of SHP1 enzymatic activity in this process. Our observations in this study indicate that SHP1 promotes tumorigenesis by a mechanism or mechanisms apart from enchancing angiogenesis. In addition, we have found no evidence that the overexpression of SHP1 could affect metastatic potential in the MDA-MB231 cells. ^ In the MDA-MB231 cells stably transfected with either wt or dn SHP1 the peak level of EGFR tyrosine phosphorylation induced by EGF, as well as the sensitivity to EGF stimulation, was not altered. However, the overexpression of wt SHP1 led to a slight increase in the kinetics of EGFR dephosphorylation, whereas the overexpression of dn SHP1 led to slightly delayed kinetics of EGFR dephosphorylation. The overexpression of either the wt or dn SHP1 did not lead to any significant increase in Src kinase activity. ^ In NIH3T3 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by EGF or Akt activation by PDGF. In 3T3H4 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by heregulin. The transient overexpression of wt SHP1 in the MDA-MB231 cells caused an apparent increase, ranging from 10% to 20%, in the G0/G1 population of the cells with a corresponding decrease in the S phase population. ^ In order to understand the mechanisms by which SHP1 exerts its positive effect on the tumorigenic potential of the MDA-MB231 cells, we employed two-dimensional electrophoresis in an attempt to identify cellular protein(s) with significantly altered tyrosine phosphorylation level upon wt SHP1 overexpression. The overexpression of wt SHP1 but not dn SHP1, leads increased tyrosine phosphorylation of a protein with a molecular weight of approximately 40 kDa and a pI between 5.9 to 6.6. ^