Piperlongumine derived cyclic sulfonamides (sultams): Synthesis and in vitro exploration for therapeutic potential against HeLa cancer cell lines.

A novel modification of piperlongumine is designed, bearing a cyclic sulphonamide (sultam) and its synthesis is described. For the first time herein we report the synthesis and biological evaluation of the natural product derived cyclic sulfonamides using Grubbs second generation catalyst (Grubbs II) via ring closing metathesis approach. Synthesis of a series of piperlongumine derived sultams is done in a moderate to good yield using Wittig reaction, Ring-Closing Metathesis (RCM) and, amide synthesis by using mixed anhydride, approach. All synthesized compounds were evaluated for anticancer activity and some demonstrated dose dependent reduction in HeLa cell growth. Of these 7, 10 and 14 significantly reduced the cell growth. Consequently their calculated GI50 values were found to be 0.1 or <0.1 µM.

Structural basis of functional group activation by sulfotransferases in complex metabolic pathways.

Sulfated molecules with diverse functions are common in biology, but sulfonation as a method to activate a metabolite for chemical catalysis is rare. Catalytic activity was characterized and crystal structures were determined for two such "activating" sulfotransferases (STs) that sulfonate ß-hydroxyacyl thioester substrates. The CurM polyketide synthase (PKS) ST domain from the curacin A biosynthetic pathway of Moorea producens and the olefin synthase (OLS) ST from a hydrocarbon-producing system of Synechococcus PCC 7002 both occur as a unique acyl carrier protein (ACP), ST, and thioesterase (TE) tridomain within a larger polypeptide. During pathway termination, these cyanobacterial systems introduce a terminal double bond into the ß-hydroxyacyl-ACP-linked substrate by the combined action of the ST and TE. Under in vitro conditions, CurM PKS ST and OLS ST acted on ß-hydroxy fatty acyl-ACP substrates; however, OLS ST was not reactive toward analogues of the natural PKS ST substrate bearing a C5-methoxy substituent. The crystal structures of CurM ST and OLS ST revealed that they are members of a distinct protein family relative to other prokaryotic and eukaryotic sulfotransferases. A common binding site for the sulfonate donor 3'-phosphoadenosine-5'-phosphosulfate was visualized in complexes with the product 3'-phosphoadenosine-5'-phosphate. Critical functions for several conserved amino acids in the active site were confirmed by site-directed mutagenesis, including a proposed glutamate catalytic base. A dynamic active-site flap unique to the "activating" ST family affects substrate selectivity and product formation, based on the activities of chimeras of the PKS and OLS STs with exchanged active-site flaps.

Structure-based design, synthesis, and biological evaluation of dihydroquinazoline-derived potent ß-secretase inhibitors.

Structure-based design, synthesis, and biological evaluation of a series of dihydroquinazoline-derived ß-secretase inhibitors incorporating thiazole and pyrazole-derived P2-ligands are described. We have identified inhibitor 4f which has shown potent enzyme inhibitory (K(i)=13 nM) and cellular (IC(50)=21 nM in neuroblastoma cells) assays. A model of 4f was created based upon the X-ray structure of 3a-bound ß-secretase. The model suggested possible interactions in the active site.

Immature T-cell clustering and efficient differentiation require the polarity protein Scribble.

T-cell polarization is required for cell migration and cell-cell interactions, cellular behaviors crucial for lymphocyte differentiation. Despite expression of the epithelial polarity network in T cells, neither its contribution to thymocyte polarity nor its requirement during development is known. We report here that depletion of the polarity protein Scribble in hematopoietic progenitor cells results in inefficient T-cell development characterized by a partial developmental block during the early double-negative (DN) stage of differentiation. Scribble-depleted hematopoietic progenitor cells exhibit a delayed transition into late CD44(lo/-)CD25(+) DN3 cells, evidenced by the accumulation of early CD44(int)CD25(+) DN3 cells. As a consequence, a limited cellular expansion and a reduced frequency of intracellular T-cell receptor ß-positive DN3 cells are observed among Scribble-deficient differentiating T cells. Moreover, whereas purified Scribble-depleted DN2 and DN3 cells do not exhibit compromised spontaneous motility, T-cell clustering and prolonged homotypic interactions among such cells are reduced. This deficiency correlates with a lack of polarization of the integrin LFA-1 during T-cell migration or on the initiation of T-cell-T-cell interactions. Scribble is therefore a critical contributor to the clustering of immature T cells, an event shown here to be necessary for efficient developmental progression.

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-imidazolide alters transforming growth factor beta-dependent signaling and cell migration by affecting the cytoskeleton and the polarity complex.

The anti-tumor synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-imidazolide (CDDO-Im) ectopically activates the transforming growth factor beta (TGFbeta)-Smad pathway and extends the duration of signaling by an undefined mechanism. Here we show that CDDO-Imdependent persistence of Smad2 phosphorylation is independent of Smad2 phosphatase activity and correlates with delayed TGFbeta receptor degradation and trafficking. Altered TGFbeta trafficking parallels the dispersal of EEA1-positive endosomes from the perinuclear region of CDDO-Im-treated cells. The effect of CDDO-Im on the EEA1 compartment led to an analysis of the cytoskeleton, and we observed that CDDO-Im alters microtubule dynamics by disrupting the microtubule-capping protein, Clip-170. Interestingly, biotinylated triterpenoid was found to localize to the polarity complex at the leading edge of migrating cells. Furthermore, CDDO-Im disrupted the localization of IQGAP1, PKCzeta, Par6, and TGFbeta receptors from the leading edge of migrating cells and inhibited TGFbeta-dependent cell migration. Thus, the synthetic triterpenoid CDDO-Im interferes with TGFbeta receptor trafficking and turnover and disrupts cell migration by severing the link between members of the polarity complex and the microtubule network.

A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells.

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Plk4 haploinsufficiency causes mitotic infidelity and carcinogenesis.

The polo-like kinase Plk4 (also called Sak) is required for late mitotic progression, cell survival and postgastrulation embryonic development. Here we identified a phenotype resulting from Plk4 haploinsufficiency in Plk4 heterozygous cells and mice. Plk4+/- embryonic fibroblasts had increased centrosomal amplification, multipolar spindle formation and aneuploidy compared with wild-type cells. The incidence of spontaneous liver and lung cancers was approximately 15 times high in elderly Plk4+/- mice than in Plk4+/+ littermates. Using the in vivo model of partial hepatectomy to induce synchronous cell cycle entry, we determined that the precise regulation of cyclins D1, E and B1 and of Cdk1 was impaired in Plk4+/- regenerating liver, and p53 activation and p21 and BubR1 expression were suppressed. These defects were associated with progressive cell cycle delays, increased spindle irregularities and accelerated hepatocellular carcinogenesis in Plk4+/- mice. Loss of heterozygosity occurs frequently (approximately 60%) at polymorphic markers adjacent to the PLK4 locus in human hepatoma. Reduced Plk4 gene dosage increases the probability of mitotic errors and cancer development.

The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis.

Rho GTPases regulate reorganization of actin and microtubule cytoskeletal structures during both interphase and mitosis. The timing and subcellular compartment in which Rho GTPases are activated is controlled by the large family of Rho GTP exchange factors (RhoGEFs). Here, we show that the microtubule-associated RhoGEF Lfc is required for the formation of the mitotic spindle during prophase/prometaphase. The inability of cells to assemble a functioning spindle after Lfc inhibition resulted in a delay in mitosis and an accumulation of prometaphase cells. Inhibition of Lfc's primary target Rho GTPase during prophase/prometaphase, or expression of a catalytically inactive mutant of Lfc, also prevented normal spindle assembly and resulted in delays in mitotic progression. Coinjection of constitutively active Rho GTPase rescued the spindle defects caused by Lfc inhibition, suggesting the requirement of RhoGTP in regulating spindle assembly. Lastly, we implicate mDia1 as an important effector of Lfc signaling. These findings demonstrate a role for Lfc, Rho, and mDia1 during mitosis.

TMF/ARA160 is a BC-box-containing protein that mediates the degradation of Stat3.

TMF/ARA160 is a Golgi resident protein whose cellular functions have not been conclusively revealed. Herein we show that TMF/ARA160 can direct the proteasomal degradation of the key cell growth regulator - Stat3. TMF/ARA160 was dispersed in the cytoplasm of myogenic C2C12 cells that were grown under low-serum conditions. The cytoplasmic distribution of TMF/ARA160 was accompanied by its transient association with the tyrosine kinase Fer and with Stat3, which underwent proteasomal degradation under those conditions. Moreover, serum deprivation induced the association of ubiquitinated proteins, with the TMF/ARA160 complex. However, TMF/ARA160 did not bind Stat1, whose cellular levels were increased in serum-starved C2C12 cells. Amino-acid sequence analysis identified a BC-box element in TMF/ARA160 that mediated the binding of this protein to elongin C. Ectopic expression of TMF/ARA160 in serum-starved C2C12 cells drove the ubiquitination and proteasomal degradation of Stat3, an effect that was not caused by TMF/ARA160 devoid of the BC-box motif. Thus, the Golgi apparatus harbors a novel BC-box-containing protein that can direct Stat3 to proteasomal degradation. Interestingly, the level of TMF/ARA160 was significantly decreased in malignant brain tumors, implying a suppressive role of that protein in tumor progression.

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

A polarity complex of mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl.

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.